Method and system for the analysis of saliva using a sensor array

ABSTRACT

A system for the rapid characterization of analytes in saliva. In one embodiment, a system for detecting analytes includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member, in which a plurality of cavities may be formed. A series of chemically sensitive particles, in one embodiment, are positioned within the cavities. The particles may produce a signal when a receptor, coupled to the particle, interacts with the cardiovascular risk factor analyte and the particle-analyte complex is visualized using a visualization reagent. Using pattern recognition techniques, the analytes within a multi-analyte fluid may be characterized. In an embodiment, each cavity of the plurality of cavities is designed to capture and contain a specific size particle. Flexible projections may be positioned over each of the cavities to provide retention of the particles in the cavities.

PRIORITY CLAIM

This application claims priority to U.S. provisional patent applicationNo. 60/528,946 entitled “Method and System for the Analysis of SalivaUsing a Sensor Array” to McDevitt et al. filed on Dec. 11, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and device for the analysis ofanalytes in saliva. More particularly, the invention relates to thedevelopment and use of a sensor array system capable of discriminatingmultiple analytes in saliva.

2. Brief Description of the Related Art

Interest in saliva as a diagnostic medium has increased exponentially inthe last 10 years. In the United States, the need for further researchin salivary diagnostics has been emphasized by federal action plansoriginating from the Office of the Surgeon General (Health and HumanServices (HHS), 2000) and the National Institute of Dental andCraniofacial Research, (NIDCR, 1999). It is becoming increasinglyimportant to have the ability to measure relevant markers in saliva inorder to first, identify the presence of disease and, second, to monitorthe progress of the affected individual undergoing treatment. Eventhough saliva offers advantages in diagnosis by being easily accessiblethrough non-invasive collection, it presents the challenge of havingrelevant markers of disease at much lower concentrations than blood. Inaddition, the viscous nature of the saliva matrix introduces a physicalbarrier to the development of saliva-specific assays, especially forthose utilizing automated fluid delivery systems. Such problems may onlybe resolved with a significant dilution of the saliva sample, whichconsequently requires the diagnostic test to be very sensitive to beeffective for saliva samples.

Screening for cardiovascular disease is one area in which measurementsof saliva markers may be useful. Current screening and managementstrategies for risk assessment for the development of heart diseasetarget blood-based factors as predictors of cardiovascular risk. Some ofthese important factors may indeed be present in saliva but,unfortunately, most of the methods currently used for their measurementare rather inefficient. These tests require long assays, sophisticatedinstrumentation, and significant amounts of expensive reagents.Furthermore, these methods are limited to measuring just one factor at atime and, in most cases, they are not sensitive enough to detect thoserelevant markers present in saliva at such low concentrations.

SUMMARY OF THE INVENTION

Herein we describe systems and methods for the analysis of a salivacontaining one or more analytes of interest. In one embodiment, theanalytes of interest include cardiac risk factors. The system, in someembodiments, may generate patterns that are diagnostic for bothindividual analytes and mixtures of analytes. The system, in someembodiments, includes a plurality of chemically sensitive particles,formed in an ordered array, capable of simultaneously detecting manydifferent kinds of analytes rapidly. An aspect of the system may beforming the array using microfabrication processing, thus allowing thesystem to be manufactured in an inexpensive manner.

In an embodiment of a system for detecting analytes, the system, in someembodiments, includes a light source, a sensor array, and a detector.The sensor array, in some embodiments, is formed of a supporting memberformed to hold a variety of chemically sensitive particles (hereinreferred to as “particles”) in an ordered array. The particles are, insome embodiments, elements, which will create a detectable signal in thepresence of an analyte. The particles may produce optical (e.g.,absorbance or reflectance) or fluorescence/phosphorescent signals uponexposure to an analyte. A detector (e.g., a charge-coupled device,“CCD”), in one embodiment, is positioned below the sensor array to allowfor data acquisition. In another embodiment, the detector may bepositioned above the sensor array to allow for data acquisition fromreflectance of light off particles.

Light originating from the light source may pass through the sensorarray and out through the bottom side of the sensor array. Lightmodulated by the particles may pass through the sensor array and ontothe proximally spaced detector. Evaluation of the optical changes may becompleted by visual inspection or by use of a CCD detector by itself orin combination with an optical microscope. A microprocessor may becoupled to the CCD detector or the microscope. A fluid delivery systemmay be coupled to the supporting member of the sensor array. The fluiddelivery system, in some embodiments, introduces samples into and out ofthe sensor array.

In an embodiment, a sensor array system includes an array of particles.The particles may include a receptor molecule coupled to a polymericbead. The receptors, in some embodiments, are chosen for interactingwith analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles.The supporting member may allow the passage of the appropriatewavelengths of light. Light may pass through all of, or a portion of,the supporting member. The supporting member may include a plurality ofcavities. The cavities may be formed such that at least one particle issubstantially contained within the cavity.

In an embodiment, an optical detector may be integrated within thebottom of the supporting member, rather than using a separate detectingdevice. The optical detectors may be coupled to a microprocessor toallow evaluation of fluids without the use of separate detectingcomponents. Additionally, a fluid delivery system may also beincorporated into the supporting member. Integration of detectors and afluid delivery system into the supporting member may allow the formationof a compact and portable analyte sensing system.

A high sensitivity CCD array may be used to measure changes in opticalcharacteristics, which occur upon binding of biological/chemical agents.The CCD arrays may be interfaced with filters, light sources, fluiddelivery, and/or micromachined particle receptacles to create afunctional sensor array. Data acquisition and handling may be performedwith existing CCD technology. CCD detectors may be used to measure whitelight, ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photo diodes,photodiode arrays, and microchannel plates may also be used.

In an embodiment, the sensor array system includes an array ofparticles. The particles may include a receptor molecule coupled to apolymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles.The supporting member may allow the passage of the appropriatewavelengths of light. Light may pass through all of or portions of thesupporting member. The supporting member may include a plurality ofcavities. The cavities may be formed such that at least one particle issubstantially contained within the cavity. A vacuum may be coupled tothe cavities. The vacuum may be applied to the entire sensor array.Alternatively, a vacuum apparatus may be coupled to the cavities toprovide a vacuum to the cavities. A vacuum apparatus is any devicecapable of creating a pressure differential to cause fluid movement. Thevacuum apparatus may apply a pulling force to any fluids within thecavity. The vacuum apparatus may pull the fluid through the cavity.Examples of vacuum apparatuses include a pre-sealed vacuum chamber,vacuum pumps, vacuum lines, or aspirator-type pumps.

A particle, in some embodiments, may possess both the ability to bindthe analyte of interest and to create a modulated signal. The particlemay include receptor molecules which posses the ability to bind theanalyte of interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte of interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal.

A variety of natural and synthetic receptors may be used. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. In one embodiment, a naturallyoccurring or synthetic receptor is bound to a polymeric bead in order tocreate the particle. The particle, in some embodiments, is capable ofboth binding the analyte(s) of interest and creating a detectablesignal. In some embodiments, the particle will create an optical signalwhen bound to an analyte of interest. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner.

The synthetic receptors may come from a variety of classes including,but not limited to, polynucleotides (e.g., aptamers), peptides (e.g.,enzymes and antibodies), synthetic receptors, polymeric unnaturalbiopolymers (e.g., polythioureas, polyguanidiniums), and imprintedpolymers. Polynucleotides are relatively small fragments of DNA, whichmay be derived by sequentially building the DNA sequence. Peptides mayinclude natural peptides, such as antibodies or enzymes or synthesizedfrom amino acids. Unnatural biopolymers are chemical structures whichare based on natural biopolymers, but which are built from unnaturallinking units. For example, polythioureas and polyguanidiniums may besynthesized from diamines (i.e., compounds that include at least twoamine functional groups) rather than amino acids and have a structuresimilar to peptides. Synthetic receptors are designed organic orinorganic structures capable of binding various analytes.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors. Bacteria may also be detected using asimilar system. To detect, sense, and identify intact bacteria, the cellsurface of one bacterium may be differentiated from other bacteria, orgenomic material may be detected using oligonucleic receptors. Onemethod of accomplishing this differentiation is to target cell surfaceoligosaccharides (i.e., sugar residues). Synthetic receptors, which arespecific for oligosaccharides, may be used to determine the presence ofspecific bacteria by analyzing for cell surface oligosaccharides.

In one embodiment, a receptor may be coupled to a polymeric resin. Thereceptor may undergo a chemical reaction in the presence of an analytesuch that a signal is produced. Indicators may be coupled to thereceptor or the polymeric bead. The chemical reaction of the analytewith the receptor may cause a change in the local microenvironment ofthe indicator to alter the spectroscopic properties of the indicator.The signal may be produced using a variety of signaling protocols. Suchprotocols may include absorbance, fluorescence resonance energytransfer, and/or fluorescence quenching. Receptor-analyte combinationsmay include, but are not limited to, peptides-proteases,polynucleotides-nucleases, and oligosaccharides-oligosaccharide cleavingagents.

In one embodiment, a receptor and an indicator may be coupled to apolymeric resin. The receptor may undergo a conformational change in thepresence of an analyte such that a change in the local microenvironmentof the indicator occurs. This change may alter the spectroscopicproperties of the indicator. The interaction of the receptor with theindicator may be produce a variety of different signals depending on thesignaling protocol used. Such protocols may include absorbance,fluorescence resonance energy transfer, and/or fluorescence quenching.

In an embodiment, a receptor may be coupled to a polymeric resin. Thereceptor may interact with the analyte to form a particle-analytecomplex. A visualization reagent may be applied to the particle-analytecomplex, which may produce a variety of different signals depending onthe signaling protocol used. The visualization of the complex mayinclude addition of dyes, stains or may include fluorescence resonanceenergy transfer, absorbance, and/or fluorescence quenching.

BRIEF DESCRIPTION OF THE DRAWINGS

Features and advantages of the methods and apparatus of the presentinvention will be more fully appreciated by reference to the followingdetailed description of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings in which:

FIG. 1 depicts an embodiment of an analyte detection system, whichincludes a sensor array disposed within a chamber;

FIG. 2 depicts an embodiment of an integrated analyte detection system;

FIG. 3 depicts an embodiment of a sensor array system of across-sectional view of a cavity covered by a mesh cover;

FIG. 4 depicts a top view of a cavity covered by a mesh cover of anembodiment of a sensor array system;

FIG. 5 depicts an embodiment of a sensor array;

FIG. 6 depicts a cross-sectional view of an embodiment of a sensorarray, which includes a micropump;

FIG. 7 depicts a cross-sectional view of an embodiment of a sensorarray, which includes a micropump and channels, which are coupled to thecavities;

FIG. 8 depicts a cross-sectional view of an embodiment of a sensorarray, which includes multiple micropumps, each micropump being coupledto a cavity;

FIG. 9 depicts a cross-sectional view of an embodiment of a sensorarray, which includes a system for delivering a reagent from a reagentparticle to a sensing cavity;

FIG. 10 depicts a schematic of an embodiment of an analyte detectionsystem;

FIG. 11 depicts a cross-sectional view of an embodiment of a sensorarray, which includes a vacuum chamber;

FIG. 12 depicts a cross-sectional view of an embodiment of a sensorarray, which includes a vacuum chamber, a filter, and a reagentreservoir;

FIGS. 13A-D depicts a general scheme for the testing of an antibodyanalyte of an embodiment of a sensor array system;

FIGS. 14A-D depicts a general scheme for the detection of antibodies, ofan embodiment of a sensor array composed of four individual beads;

FIG. 15 depicts an embodiment of a sensor array which includes a vacuumchamber, a sensor array chamber, and a sampling device;

FIG. 16 depicts a flow path of a fluid stream through a sensor arrayfrom the top toward the bottom of the sensor array in an embodiment of asensor array system;

FIG. 17 depicts a flow path of a fluid stream through a sensor arrayfrom the bottom toward the top of the sensor array in an embodiment of asensor array system;

FIG. 18 depicts an embodiment of a portable sensor array system;

FIGS. 19A-B depict views of an embodiment of an alternate portablesensor array;

FIG. 20 depicts an exploded view of a cartridge for use in an embodimentof a portable sensor array;

FIG. 21 depicts a cross sectional view of a cartridge for use in anembodiment of a portable sensor array;

FIG. 22 depicts the chemical constituents of a particle in an embodimentof a sensor array system;

FIG. 23 depicts a schematic view of the transfer of energy from a firstindicator to a second indicator in the presence of an analyte in anembodiment of a sensor array system;

FIGS. 24A-I depict various sensing protocols forreceptor-indicator-polymeric resin particles in an embodiment of asensor array system;

FIG. 25 depicts receptors in an embodiment of a sensor array system;

FIG. 26 depicts the attachment of differentially protected lysine to abead in an embodiment of a sensor array system;

FIG. 27 depicts a system for measuring the absorbance or emission of asensing particle;

FIG. 28 depicts receptors in an embodiment of a sensor array system;

FIG. 29 depicts pH indicators, which may be coupled to a particle in anembodiment of a sensor array system;

FIG. 30 depicts the change in FRET between coumarin and5-carboxyfluorescein on resin beads as a function of the solvent in anembodiment of a sensor array system;

FIGS. 31A-D depict various sensing protocols forreceptor-indicator-polymeric resin particles in which a cleavagereaction occurs in an embodiment of a sensor array system;

FIG. 32 depicts a graph of various diluents compared to detection levelof CRP;

FIG. 33 depicts a sensor array dose response curve for a CRP assay insaliva testing;

FIG. 34 depicts a graph of the sensitivity of an ELISA test with respectto concentration of CRP;

FIGS. 35A-B depict graphs of the comparison of a sensor array detectiondevice and ELISA methods;

FIG. 36 depicts saliva CRP levels for different types of subjects;

FIG. 37 depicts a graph of the saliva CRP levels for different types ofsubjects;

FIGS. 38A-B depict the correlation between CRP levels in saliva andserum;

FIGS. 39A-B depict the detection of Hepatitis B HbsAg in the presence ofHIV gp41/120 and Influenza A in an embodiment of a sensor array system;

FIG. 40 depicts the detection of CRP in an embodiment of a sensor arraysystem;

FIG. 41 depicts the dosage response of CRP levels in an embodiment of asensor array system;

FIGS. 42A-D depict the multi-analyte detection of CRP and IL-6 in anembodiment of a sensor array system; and

FIG. 43 depicts the regeneration of receptor particles in an embodimentof a sensor array system.

DETAILED DESCRIPTION OF EMBODIMENTS

Herein we describe a system and method for the simultaneous analysis ofa fluid containing multiple analytes. The system may generate patternsthat are diagnostic for both individual analytes and mixtures of theanalytes. The system, in some embodiments, is made of a combination ofchemically sensitive particles, formed in an ordered array, capable ofsimultaneously detecting many different kinds of analytes in salivarapidly. An aspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

Various systems for detecting analytes in a fluid and gases have beendescribed in U.S. Pat. Nos. 6,045,579; 6,680,206; 6,649,403; and6,713,298; U.S. Patent Application Publication Nos. US 2002-0197622 A1,US 2003-0064422 A1; US 2004-0053322 A1; and US 2003-0186228 A1; and inU.S. patent application Ser. No. 09/287,248 all of which areincorporated by reference as if fully set forth herein.

Shown in FIG. 1 is an embodiment of a system for detecting analytes in afluid. In one embodiment, the system includes light source 100, sensorarray 120, chamber 140 for supporting the sensor array, and detector160. Sensor array 120 may include a supporting member, which is formedto hold a variety of particles. In one embodiment, light originatingfrom light source 100 passes through sensor array 120 and out throughthe bottom side of the sensor array. Light modulated by the particlesmay be detected by proximally spaced detector 160. While depicted asbeing positioned below the sensor array, it should be understood thatthe detector might be positioned above the sensor array for reflectancemeasurements. Evaluation of the optical changes may be completed byvisual inspection (e.g., by eye, or with the aid of a microscope) or byuse of microprocessor 180 coupled to the detector.

In this embodiment, sensor array 120 is positioned within chamber 140.Chamber 140, may allow a fluid stream to pass through the chamber suchthat the fluid stream interacts with sensor array 120. The chamber maybe constructed of glass (e.g., borosilicate glass or quartz) or aplastic material transparent to a portion of the light from the lightsource. The material should also be substantially unreactive toward thefluid. Examples of plastic materials which may be used to form thechamber include, but are not limited to, acrylic resins, polycarbonates,polyester resins, polyethylenes, polyimides, polyvinyl polymers (e.g.,polyvinyl chloride, polyvinyl acetate, polyvinyl dichloride, polyvinylfluoride, etc.), polystyrenes, polypropylenes, polytetrafluoroethylenes,and polyurethanes. An example of such a chamber is a Sykes-Moorechamber, which is commercially available from Bellco Glass, Inc., NJ.

Chamber 140, in one embodiment, includes fluid inlet port 200 and fluidoutlet port 220. Fluid inlet 200 and outlet 220 ports allow a fluidstream to pass into interior 240 of the chamber during use. The inletand outlet ports may allow facile placement of a conduit fortransferring the fluid to the chamber. In one embodiment, the ports arehollow conduits. The hollow conduits may have an outer diametersubstantially equal to the inner diameter of a tube for transferring thefluid to or away from the chamber. For example, if a plastic or rubbertube is used for the transfer of the fluid, the internal diameter of theplastic tube is substantially equal to the outer diameter of the inletand outlet ports.

In another embodiment, the inlet and outlet ports may be Luer lock styleconnectors. The inlet and outlet ports may be female Luer lockconnectors. The use of female Luer lock connectors will allow a fluid tobe introduced via a syringe. Typically, syringes include a male Luerlock connector at the dispensing end of the syringe. For theintroduction of liquid samples, the use of Luer lock connectors mayallow samples to be transferred directly from a syringe to chamber 140.Luer lock connectors may also allow plastic or rubber tubing to beconnected to the chamber using Luer lock tubing connectors.

The chamber may substantially confine the fluid passage to interior 240of the chamber. By confining the fluid to a small interior volume, theamount of fluid required for an analysis may be minimized. The interiorvolume may be specifically modified for a desired application. Forexample, for the analysis of small volumes of fluid samples, the chambermay be designed to have a small interior chamber, thus reducing theamount of fluid needed to fill the chamber. For larger samples, a largerinterior chamber may be used. Larger chambers may allow a fasterthroughput of the fluid during use.

In another embodiment, depicted in FIG. 2, a system for detectinganalytes in a fluid includes light source 100, sensor array 120, chamber140 for supporting the sensor array, and detector 160 , all enclosedwithin detection system enclosure 260. As described above, sensor array120 may be formed of a supporting member to hold a variety of particles.Thus, in a single enclosure, all of the components of the analytedetection system may be included.

The formation of an analyte detection system in a single enclosure mayallow the formation of a portable detection system. For example,controller 280 may be coupled to the analyte detection system.Controller 280 may interact with the detector and display the resultsfrom the analysis. In one embodiment, the controller includes displaydevice 300 for displaying information to a user. The controller may alsoinclude input devices 320 (e.g., buttons) to allow the user to controlthe operation of the analyte detection system. The controller maycontrol operation of light source 100 and operation of detector 160.

Detection system enclosure 260 may be interchangeable with thecontroller. Coupling members 340 and 360 may be used to remove detectionsystem enclosure 260 from controller 280. A second detection systemenclosure may be readily coupled to the controller using couplingmembers 340 and 360. In this manner, a variety of different types ofanalytes may be detecting using a variety of different detection systemenclosures. Each of the detection system enclosures may includedifferent sensor arrays mounted within their chambers. Instead of havingto exchange the sensor array for different types of analysis, the entiredetection system enclosure may be exchanged. This may prove advantageouswhen a variety of detection schemes is used.

For example, a first detection system enclosure may be used for whitelight applications. The first detection system enclosure may include awhite light source, a sensor that includes particles that produce avisible light response in the presence of an analyte, and a detectorsensitive to white light. A second detection system enclosure may beused for fluorescent applications, including a fluorescent light source,a sensor array that includes particles, which produce a fluorescentresponse in the presence of an analyte, and a fluorescent detector. Thesecond detection system enclosure may also include other componentsnecessary for the detection system. For example, the second detectionsystem may also include a filter for preventing short wavelengthexcitation from producing “false” signals in the optical detectionsystem during fluorescence measurements. A user need only select theproper detection system enclosure for detection of the desired analyte.Since each detection system enclosure includes many of the requiredcomponents, a user does not have to make light source selections, sensorarray selections or detector arrangement selections to produce a viabledetection system.

In another embodiment, the individual components of the system may beinterchangeable. The system may include coupling members 380 and 400that allow light source 100 and detector 160, respectively, to beremoved from chamber 140. This may allow a modular design of the system.For example, an analysis may be first performed with a white lightsource to give data corresponding to an absorbance/reflectance analysis.The light source may then be changed to an ultraviolet light source toallow ultraviolet analysis of the particles. Since the particles havealready been treated with the fluid, the analysis may be preformedwithout further treatment of the particles with a fluid. In this manner,a variety of tests may be performed using a single sensor array.

In an embodiment, a supporting member is made of any material capable ofsupporting the particles while allowing passage of an appropriatewavelength of light. The supporting member may also be made of amaterial substantially impervious to the fluid in which the analyte ispresent. A variety of materials may be used including plastics (e.g.,photoresist materials, acrylic polymers, carbonate polymers, etc.),glass, silicon based materials (e.g., silicon, silicon dioxide, siliconnitride, etc.) and metals.

In one embodiment, the supporting member includes a plurality ofcavities. Each cavity may be formed such that at least one particle issubstantially contained within the cavity. In another embodiment, aplurality of particles may be contained within a single cavity.

In some embodiments, it may be necessary to pass liquids over the sensorarray. The dynamic motion of liquids across the sensor array may lead todisplacement of the particles from the cavities. In another embodiment,the particles may be held within cavities formed in a supporting memberby the use of a transmission electron microscope (“TEM”) grid. Asdepicted in FIG. 3, cavity 420 is formed in supporting member 440. Afterplacement of particle 460 within the cavity, TEM grid 480 may be placedatop supporting member 440 and secured into position. TEM grids andadhesives for securing TEM grids to a support are commercially availablefrom Ted Pella, Inc., Redding, Calif. TEM grid 480 may be made from anumber of materials including, but not limited to, copper, nickel, gold,silver, aluminum, molybdenum, titanium, nylon, beryllium, carbon, andberyllium-copper. The mesh structure of the TEM grid may allow solutionaccess as well as optical access to the particles that are placed in thecavities. FIG. 4 further depicts a top view of a sensor array with TEMgrid 480 secured to the upper surface of supporting member 440. TEM grid480 may be placed on the upper surface of the supporting member to trapparticles 460 within cavities 420. As depicted, openings 500 in TEM grid480 may be sized to hold particles 460 within cavities 420, whileallowing fluid and optical access cavities 420.

In another embodiment, a sensor array includes a supporting memberformed to support the particles while allowing passage of an appropriatewavelength of light to the particles. The supporting member, in oneembodiment, includes a plurality of cavities. The cavities may be formedsuch that at least one particle is substantially contained within eachcavity. The supporting member may be formed to substantially inhibit thedisplacement of particles from the cavities during use. The supportingmember may also allow passage of fluid through the cavities. The fluidmay flow from a top surface of the supporting member, past a particle,and out a bottom surface of the supporting member. This may increase thecontact time between a particle and the fluid.

Formation of a silicon based supporting member which includes aremovable top cover and bottom cover are described in U.S. patentapplication Ser. No. 09/287,248; U.S. Pat. Nos. 6,680,206; 6,649,403;and 6,713,298; and U.S. Patent Application Publication Nos. US2003-0064422 A1; US 2004-0053322 A1; US 2003-0186228 A1; and US2002-0197622 A1, which are incorporated by reference as if fully setforth herein.

In one embodiment, series of channels 520 may be formed in supportingmember 440 interconnecting at least some of cavities 420, as depicted inFIG. 5. Pumps and valves may also be incorporated into supporting member440 to aid passage of the fluid through the cavities. Pumps and valvesare described in U.S. Patent Application Publication No. US 2002-0197622A1 which is incorporated by reference as if fully set forth herein.

An advantage of using pumps may be better flow through the channel. Thechannel and cavities may have a small volume. The small volume of thecavity 420 and channel 520 tends to inhibit flow of fluid through thecavity. By incorporating pump 540, the flow of fluid to the cavity 420and through the cavity may be increased, allowing more rapid testing ofa fluid sample. While a diaphragm based pump system is depicted in FIG.6, it should be understood that electrode based pumping systems mightalso be incorporated into the sensor array to produce fluid flows.

In another embodiment, a pump may be coupled to a supporting member foranalyzing analytes in a fluid stream, as depicted in FIG. 7. Channel 520may couple pump 540 to multiple cavities 420 formed in supporting member440. Cavities 420 may include sensing particles 460. Pump 540 may createa flow of fluid through channel 520 to cavities 420. In one embodiment,cavities 420 may inhibit the flow of the fluid through the cavities. Thefluid may flow into cavities 420 and past particle 460 to create a flowof fluid through the sensor array system. In this manner, a single pumpmay be used to pass the fluid to multiple cavities. While a diaphragmpump system is depicted in FIG. 7, it should be understood thatelectrode pumping systems might also be incorporated into the supportingmember to create similar fluid flows.

In another embodiment, multiple pumps may be coupled to a supportingmember of a sensor array system. The pumps may be coupled in series witheach other to pump fluid to each of the cavities. As depicted in FIG. 8,first pump 540 and second pump 560 are coupled to supporting member 440.First pump 540 may be coupled to first cavity 420. The first pump maytransfer fluid to first cavity 420 during use. Cavity 420 may allowfluid to pass through the cavity to first cavity outlet channel 580.Second pump 560 may also be coupled to supporting member 440. Secondpump 560 may be coupled to second cavity 600 and first cavity outletchannel 580. Second pump 560 may transfer fluid from first cavity outletchannel 580 to second cavity 600. The pumps may be synchronized suchthat a steady flow of fluid through the cavities is obtained. Additionalpumps may be coupled to second cavity outlet channel 620 such that thefluid may be pumped to additional cavities. In one embodiment, each ofthe cavities in the supporting member is coupled to a pump used to pumpthe fluid stream to the cavity.

In some instances, it may be necessary to add a reagent to a particlebefore, during, or after an analysis process. Reagents may includereceptor molecules or indicator molecules. Typically, such reagents areadded by passing a fluid stream, which includes the reagent over asensor array. In an embodiment, the reagent may be incorporated into asensor array system that includes two particles. In this embodiment,sensor array system 900 may include two particles, 910 and 920, for eachsensing position of the sensor array, as depicted in FIG. 9. Firstparticle 910 may be positioned in first cavity 912. Second particle 920may be positioned in second cavity 922. In one embodiment, the secondcavity is coupled to the first cavity via channel 930. The secondparticle includes a reagent, which is at least partially removable fromthe particle. The reagent may also be used to modify first particle 910when in contacted with the first particle, such that the first particlewill produce a signal upon interaction with an analyte during use.

The reagent may be added to the first cavity before, during, or after afluid analysis. The reagent may be coupled to second particle 920. Aportion of the reagent coupled to the second particle may be decoupledfrom the particle by passing a decoupling solution past the particle.The decoupling solution may include a decoupling agent, which will causeat least a portion of the reagent to be at released from the particle.Reservoir 940 may be formed on the sensor array to hold the decouplingsolution.

First pump 950 and second pump 960 may be coupled to supporting member915. First pump 950 may be used to pump fluid from fluid inlet 952 tofirst cavity 912 via channel 930. Fluid inlet 952 may be located wherethe fluid, which includes the analyte, is introduced into the sensorarray system. Second pump 950 may be coupled to reservoir 940 and secondcavity 922. Second pump 960 may be used to transfer the decouplingsolution from the reservoir to second cavity 922. The decouplingsolution may pass through second cavity 922 and into first cavity 912.Thus, as the reagent is removed, the second particle it may betransferred to first cavity 912 where the reagent may interact withfirst particle 910. The reservoir may be filled and/or refilled byremoving reservoir outlet 942 and adding additional fluid to reservoir940. While diaphragm based pump systems are depicted in FIG. 9, itshould be understood that electrode based pumping systems might also beincorporated into the sensor array to produce fluid flows.

The use of such a system is described by way of example. In someinstances, it may be desirable to add a reagent to the first particleprior to passing a fluid to the first particle. The reagent may becoupled to the second particle and placed in the sensor array prior touse. The second particle may be placed in the array during constructionof the array. A decoupling solution may be added to the reservoir beforeuse. Controller 970, shown in FIG. 9, may also be coupled to the systemto allow automatic operation of the pumps. Controller 970 may initiatethe analysis sequence by activating second pump 960, causing thedecoupling solution to flow from reservoir 940 to second cavity 922. Asthe fluid passes through second cavity 922, the decoupling solution maycause at least some of the reagent molecules to be released from secondparticle 920. The decoupling solution may be passed out of second cavity922 and into first cavity 912. As the solution passes through the firstcavity, some of the reagent molecules may be captured by first particle910. After a sufficient number of molecules have been captured by firstparticle 910, flow of fluid thorough second cavity 922 may be stopped bycontroller 970. During initialization of the system, the flow of fluidthrough the first pump may be inhibited.

After the system is initialized, the second pump may be stopped and thefluid may be introduced to the first cavity. The first pump may be usedto transfer the fluid to the first cavity. The second pump may remainoff, thus inhibiting flow of fluid from the reservoir to the firstcavity. It should be understood that the reagent solution might be addedto the first cavity while the fluid is added to the first cavity. Inthis embodiment, both the first and second pumps may be operatedsubstantially simultaneously.

Alternatively, the reagent may be added after an analysis. In someinstances, a particle may interact with an analyte such that a change inthe receptors attached to the first particle occurs. This change,however, may not produce a detectable signal. The reagent attached tothe second particle may be used to produce a detectable signal uponinteraction with the first particle if a specific analyte is present. Inthis embodiment, the fluid is introduced into the cavity first. Afterthe analyte has been given, time to react with the particle, the reagentmay be added to the first cavity. The interaction of the reagent withthe particle may produce a detectable signal. For example, an indicatorreagent may react with a particle, which has been exposed to an analyteto produce a color change on the particle. A particle, which has notbeen exposed to the analyte may remain unchanged or show a differentcolor change.

As shown in FIG. 10, a system for detecting analytes in a fluid mayinclude light source 100, sensor array 120, detector 130, and controller970. Sensor array 120 may be formed of a supporting member 440 formed tohold a variety of particles 460 in an ordered array. A high sensitivityCCD array may be used to measure changes in optical characteristics,which occur upon binding of the biological/chemical agents. Dataacquisition and handling may be performed using existing CCD technology.As described above, colorimetric analysis may be performed using a whitelight source and a color CCD detector. However, color CCD detectors aretypically more expensive than gray scale CCD detectors.

In one embodiment, a gray scale CCD detector may be used to detectcolorimetric changes. A gray scale detector may be disposed below asensor array to measure the intensity of light being transmitted throughthe sensor array. A series of lights (e.g., light emitting diodes) maybe arranged above the sensor array. In one embodiment, groups of threeLED lights may be arranged above each of the cavities of the array. Eachof these groups of LED lights may include a red, blue, and green light.Each of the lights may be operated individually such that one of thelights may be on while the other two lights are off. In order to providecolor information while using a gray scale detector, each of the lightsis sequentially turned on and the gray scale detector is used to measurethe intensity of the light passing through the sensor array. Afterinformation from each of the lights is collected, the information may beprocessed to derive the absorption changes of the particle.

In one embodiment, data collected by the gray scale detector may berecorded using 8 bits of data. Thus, the data will appear as a valuebetween 0 and 255. The color of each chemical sensitive element may berepresented as a red, blue, and green value. For example, a blankparticle (i.e., a particle which does not include a receptor) willtypically appear white. When each of the LED lights (red, blue, andgreen) is operated, the CCD detector will record a value correspondingto the amount of light transmitted through the cavity. The intensity ofthe light may be compared to a blank particle to determine theabsorbance of a particle with respect to the LED light used. Thus, thered, green, and blue components may be recorded individually without theuse of a color CCD detector.

In one embodiment, it is found that a blank particle exhibits anabsorbance of about 253 when illuminated with a red LED, a value ofabout 250 when illuminated by a green LED, and a value of about 222 whenilluminated with a blue LED. This signifies that a blank particle doesnot significantly absorb red, green, or blue light. When a particle witha receptor is scanned, the particle may exhibit a color change due toabsorbance by the receptor. For example, when a particle including a5-carboxyfluorescein receptor is subjected to white light, the particleshows a strong absorbance of blue light. When a red LED is used toilluminate the particle, the gray scale CCD detector may detect a valueof about 254. When the green LED is used, the gray scale detector maydetect a value of about 218. When a blue LED light is used, a gray scaledetector may detect a value of about 57. The decrease in transmittanceof blue light is believed to be due to the absorbance of blue light bythe 5-carboxyfluorescein. In this manner, the color changes of aparticle may be quantitatively characterized using a gray scaledetector.

As described above, after the cavities are formed in the supportingmember, a particle may be positioned at the bottom of a cavity asdescribed in U.S. patent application Ser. No. 09/287,248; U.S. Pat. Nos.6,680,206; 6,649,403; and 6,713,298; and U.S. Patent ApplicationPublication Nos. US 2003-0064422 A1; US 2004-0053322 A1; US 2003-0186228A1; and US 2002-0197622 A1, which are incorporated by reference as iffully set forth herein. This allows the location of a particularparticle to be precisely controlled during the production of the array.

One challenge in a chemical sensor system is keeping “dead volume” to aminimum. This is especially problematic when an interface to the outsideworld is required (e.g., a tubing connection). In many cases, the “deadvolume” associated with delivery of a sample to the reaction site in a“lab-on-a-chip” may far exceed the actual amount of reagent required forthe reaction. Filtration is also frequently necessary to prevent smallflow channels in the sensor arrays from plugging. Here the filter can bemade an integral part of the sensor package.

In an embodiment, a system for detecting an analyte in a fluid includesa conduit coupled to a sensor array, and a vacuum chamber coupled to theconduit. FIG. 11 depicts a system in which fluid stream E passes throughconduit D, onto sensor array G, and into vacuum apparatus F. Vacuumapparatus F may be coupled to conduit D downstream from sensor array G.A vacuum apparatus is herein defined to be any system capable ofcreating or maintaining a volume at a pressure below atmospheric. Anexample of a vacuum apparatus is a vacuum chamber. A vacuum chamber, inone embodiment, may include sealed tubes from which a portion of air hasbeen evacuated to create a vacuum within the tube. A commonly usedexample of such a sealed tube is a “vacutainer” system commerciallyavailable from Becton Dickinson. Alternatively, a vacuum chamber sealedby a movable piston may also be used to generate a vacuum. For example,a syringe may be coupled to the conduit. Movement of the piston (i.e.,the plunger) away from the chamber will create a partial vacuum withinthe chamber. Alternatively, the vacuum apparatus maybe a vacuum pump orvacuum line. Vacuum pumps may include direct drive pumps, oil pumps,aspirator pumps, or micropumps. Micropumps that may be incorporated intoa sensor array system have been previously described.

As opposed to previously described methods, in which a pump is used toforce a fluid stream through a sensor array, the use of a vacuumapparatus allows the fluid to be pulled through the sensor array.Referring to FIG. 12, vacuum apparatus F is coupled downstream fromsensor array G. When coupled to the conduit D, the vacuum apparatus mayexert a suction force on a fluid stream, forcing a portion of the streamto pass over, and in some instances, through, sensor array G. In someembodiments, the fluid may continue to pass through conduit D afterpassing sensor array G, and into vacuum apparatus F.

In an embodiment where the vacuum apparatus is a pre-evacuated tube, thefluid flow will continue until the air within the tube is at a pressuresubstantially equivalent to atmospheric pressure. The vacuum apparatusmay include penetrable wall H. Penetrable wall H forms a seal inhibitingair from entering vacuum apparatus F. When wall H is broken orpunctured, air from outside the system will begin to enter the vacuumapparatus. In one embodiment, conduit D includes a penetrating member(e.g., a syringe needle), which allows the penetrable wall to bepierced. Piercing penetrable wall H causes air and fluid inside theconduit to be pulled through the conduit and into the vacuum apparatusuntil the pressure between vacuum apparatus F and conduit D isequalized.

The sensor array system may also include filter B coupled to conduit D,as depicted in FIG. 12. The filter B may be positioned along conduit D,upstream from sensor array G. Filter B may be a porous filter, whichincludes a membrane for removing components from the fluid stream. Inone embodiment, filter B may include a membrane for removal ofparticulates above a minimum size. The size of the particulates removedwill depend on the porosity of the membrane as is known in the art.Alternatively, the filter may be used to remove unwanted components of afluid stream. For example, if a fluid stream is a blood sample, thefilter may be used to remove red and white blood cells from the stream,leaving plasma and other components in the stream.

The sensor array may also include reagent delivery reservoir C. Reagentdelivery reservoir C may be coupled to conduit D upstream from sensorarray G. Reagent delivery reservoir C may be formed from a porousmaterial, which includes a reagent of interest. As the fluid passesthrough this reservoir, a portion of the reagent within the regentdelivery reservoir passes into the fluid stream. The fluid reservoir mayinclude a porous polymer or filter paper on which the reagent is stored.Examples of reagents which may be stored within the reagent deliveryreservoir include, but are not limited to, visualization agents (e.g.,dye or fluorophores), co-factors, buffers, acids, bases, oxidants, andreductants.

The sensor array may also include fluid sampling device A coupled toconduit D. Fluid sampling device A may be used to transfer a fluidsample from outside sensor array G to conduit D. A number of fluidsampling devices may be used, including, but not limited to, a syringeneedle, a tubing connector, a capillary tube, or a syringe adapter.

The sensor array may also include a micropump or a microvalve systemcoupled to the conduit to further aid in transfer of fluid through theconduit. Micropumps and valves are described in U.S. Patent ApplicationPublication No. US 2002-0197622 A1, which is fully incorporated herein.In one embodiment, a microvalve or micropump may be used to keep a fluidsample or a reagent solution separated from the sensor array. Typically,these microvalves and micropumps include a thin flexible diaphragm. Thediaphragm may be moved to an open position, in one embodiment, byapplying a vacuum to the outside of the diaphragm. In this way, a vacuumapparatus coupled to the sensor array may be used to open a remotemicrovalve or pump.

In another embodiment, a microvalve may be used to control theapplication of a vacuum to a system. For example, a microvalve may bepositioned adjacent to a vacuum apparatus. The activation of themicrovalve may allow the vacuum apparatus to communicate with a conduitor sensor array. The microvalve may be remotely activated at controlledtimes and for controlled intervals.

A sensor array system, such as depicted in FIG. 12, may be used foranalysis of blood samples. A micropuncture device A may be used toextract a small amount of blood from a patient, e.g., through afinger-prick. The blood may be drawn through a porous filter that servesto remove undesirable particulate matter. For the analysis of antibodiesor antigens in whole blood, a filtering agent may be chosen to removeboth white and red blood cells while leaving in the fluid stream bloodplasma and all of the components therein. Methods of filtering bloodcells from whole blood are taught, for example, in U.S. Pat. Nos.5,914,042, 5,876,605, and 5,211,850, which are incorporated byreference. The filtered blood may also be passed through a reagentdelivery reservoir including a porous layer impregnated with thereagent(s) of interest. In many cases, a visualization agent will beincluded in this layer so that the presence of the analytes of interestcan be resolved. The treated fluid may be passed above an electronictongue chip through a capillary layer, down through the various sensingparticles, and through the chip onto a bottom capillary layer. Afterexiting a central region, the excess fluid flows into the vacuumapparatus. This excess fluid may serve as a source of samples for futuremeasurements. A “hard copy” of the sample is thus created to back upelectronic data recorded for the specimen.

Other examples of procedures for testing bodily fluids are described inthe following U.S. Pat. Nos. 4,596,657; 4,189,382; 4,115,277; 3,954,623;4,753,776; 4,623,461; 4,069,017; 5,053,197; 5,503,985; 3,696,932;3,701,433; 4,036,946; 5,858,804; 4,050,898; 4,477,575; 4,810,378;5,147,606; 4,246,107; and 4,997,577, all of which are incorporated byreference.

The generally described sampling method may also be used for eitherantibody or antigen testing of bodily fluids. A general scheme fortesting antibodies is depicted in FIGS. 13A-D. FIG. 13A depicts apolymer bead having a protein coating that can be recognized in aspecific manner by a complimentary antibody. Three antibodies (shownwithin the dashed rectangle) are shown to be present in a fluid phasethat bathes the polymer bead. Turning to FIG. 13B, the complimentaryantibody binds to the bead while the other two antibodies remain in thefluid phase. A large increase in the complimentary antibodyconcentration is noted at this bead. In FIG. 13C, a visualization agentsuch as a protein (shown within the dashed rectangle) is added to thefluid phase. The visualization agent is chosen because either itpossesses a strong absorbance property or it exhibits fluorescencecharacteristics that can be used to identify the species of interest viaoptical measurements. The protein is an example of a reagent thatassociates with a common region of most antibodies. Chemicalderivatization of visualization agent with dyes, quantum particles, orfluorophores, is used to evoke desired optical characteristics. Afterbinding to the bead-localized antibodies, as depicted in FIG. 13D, thevisualization agent reveals the presence of complimentary antibodies atspecific polymer bead sites.

FIGS. 14A-D depicts another general scheme for the detection ofantibodies, which uses a sensor array composed of four individual beads.Each of the four beads is coated with a different antigen (e.g., aprotein coating). As depicted in FIG. 14A, the beads are washed with afluid sample, which includes four antibodies. Each of the fourantibodies binds to its complimentary antigen coating, as depicted inFIG. 14B. A visualization agent may be introduced into the chamber, asdepicted in FIG. 14C. The visualization agent, in one embodiment, maybind to the antibodies, as depicted in FIG. 14D. The presence of thelabeled antibodies is assayed by optical means (e.g., absorbance,reflectance, and/or fluorescence). Because the location of the antigencoatings is known ahead of time, the chemical/biochemical composition ofthe fluid phase can be determined from the pattern of optical signalsrecorded at each site.

In an alternative methodology, not depicted, the antibodies in thesample may be exposed to the visualization agent prior to theirintroduction into the chip array. This may render the visualization stepdepicted in FIG. 14C unnecessary.

FIG. 15 depicts a system for detecting an analyte in a fluid stream. Thesystem includes a vacuum apparatus, a chamber in which a sensor arraymay be disposed, and an inlet system for introducing the sample into thechamber. In this embodiment, the inlet system is depicted as amicro-puncture device. The chamber holding the sensor array may be aSikes-Moore chamber, as previously described. The vacuum apparatus is astandard “vacutainer” type vacuum tube. The micro puncture deviceincludes a Luer-lock attachment, which can receive a syringe needle.Between the micro-puncture device and the chamber, a syringe filter maybe placed to filter the sample as the sample enters the chamber.Alternatively, a reagent may be placed within the filter. The reagentmay be carried into the chamber via the fluid as the fluid passesthrough the filter.

As has been previously described, a sensor array may allow a fluidsample to pass through a sensor array during use. Fluid delivery to thesensor array may be accomplished by having the fluid enter the top ofthe chip through capillary A, as depicted in FIG. 16. The fluidtraverses the chip and exits from bottom capillary B. Between the topand bottom capillaries, the fluid passes by the particle. The fluid,containing analytes, has an opportunity to encounter receptor sites ofthe particle. The presence of analytes may be identified using opticalmeans as previously mentioned. Fluid flow in a forward direction forcesthe particle towards the bottom of the cavity. Under thesecircumstances, the particle is placed for ideal optical measurements, inview of light pathway D.

In another embodiment, fluid flow may go from the bottom of the sensorarray toward the top of the sensor array, as depicted in FIG. 17. In areverse flow direction, the fluid exits the top of the chip throughcapillary A. The fluid flow traverses the chip and enters the cavityfrom the bottom capillary B. Between the top and bottom capillaries, thefluid may avoid at least a portion of the particle by taking indirectpathway C. The presence of analytes may be identified using opticalmeans as before. Unfortunately, only a portion of the light may passthrough the particle. In the reverse flow direction, the particle may bepartially removed from the path of an analysis light beam D by an upwardpressure of the fluid, as shown in FIG. 17. Under these circumstances,some of the light may traverse the chip by path E and enter a detectorwithout passing through the sensor particle.

In any microfluidic chemical sensing system, there may be a need tostore chemically sensitive elements in an inert environment. Theparticles may be at least partially surrounded by an inert fluid, suchas an inert, non-reactive gas, a non-reactive solvent, or a liquidbuffer solution. Alternatively, the particles may be maintained under avacuum. Before exposure of the particles to an analyte, the inertenvironment may need to be removed to allow proper testing of a sampleof containing the analyte. In one embodiment, a system may include afluid transfer system for the removal of an inert fluid prior tointroduction of the sample with minimum dead volume.

In one embodiment, a pumping system may be used to pull the inert fluidthrough the array from one side of the array. The pumping system mayprovide pumping action downstream from the array. The inert fluid may beefficiently removed while the beads remain within the sensor array.Additionally, the analyte sample may be drawn toward the sensor array asthe inert fluid is being removed from the sensor array. A pocket of airmay separate the analyte sample from the inert fluid as the sample movesthrough the array. Alternatively, the sample may be pumped from anupstream micropump. A vacuum downstream may produce a maximum of aboutone atmosphere of head pressure, while an upstream pump may produce anarbitrarily high head pressure. This can affect fluid transport ratesthrough the system. For small volume microfluidic systems, even with lowflow coefficients, one atmosphere of head pressure may provideacceptable transfer rates for many applications.

In another embodiment, a vacuum apparatus may be formed directly into amicromachined array. The vacuum apparatus may transmit fluid to and froma single cavity or a plurality of cavities. In an alternate embodiment,a separate vacuum apparatus may be coupled to each of the cavities.

After the cavities are formed in the supporting member, a particle maybe positioned at the bottom of a cavity using a micromanipulator. Thisallows the location of a particular particle to be precisely controlledduring the production of the array. The use of a micromanipulator may beimpractical for mass-production of sensor arrays. A number of methodsfor inserting particles that may be amenable to an industrialapplication have been devised. Examples of micromanipulators anddispense heads are described in U.S. Patent Application Publication No.US 2002-0197622 A1, which is fully incorporated as set forth herein.

In one embodiment, the use of a micromanipulator may be automated.Particles may be “picked and placed” using a robotic automated assembly.The robotic assembly may include one or more dispense heads. A dispensehead may pick up and hold a particle. Alternatively, a dispense head mayhold a plurality of particles and dispense only a portion of the heldparticles. An advantage of using a dispense head is that individualparticles or small groups of particles may be placed at preciselocations on the sensor array. A variety of different types of dispenseheads may be used.

A sensor array system becomes most powerful when the associatedinstrumentation may be delivered and utilized at the application site.That is, rather than remotely collecting the samples and bringing themto a centrally based analysis site; it may be advantageous to be able toconduct the analysis at the testing location. Such a system may be used,for example, for point of care medicine, on site monitoring of processcontrol applications, military intelligence gathering devices,environmental monitoring, and food safety testing.

An embodiment of a portable sensor array system is depicted in FIG. 18.The portable sensor array system would have, in one embodiment, a sizeand weight that would allow the device to be easily carried by a personto a testing site. The portable sensor array system includes a lightsource, a sensor array, and a detector. The sensor array, in someembodiments, is formed on a supporting member to hold a variety ofparticles in an ordered array. The particles are, in some embodiments,elements that create a detectable signal in the presence of an analyte.The particles may include a receptor molecule coupled to a polymericbead. The receptors may be chosen for interacting with specificanalytes. This interaction may take the form of a binding/association ofthe receptors with the analytes. The supporting member may be made ofany material capable of supporting the particles. The supporting membermay include a plurality of cavities. The cavities may be formed suchthat at least one particle is substantially contained within the cavity.The sensor array has been previously described in detail.

The portable sensor array system may be used for a variety of differenttesting. The flexibility of sensor array system 1000, with respect tothe types of testing, may be achieved using a sensor array cartridge.Turning to FIG. 18, sensor array cartridge 1010 may be inserted intoportable sensor array system 1000 prior to testing. The type of sensorarray cartridge used will depend on the type of testing to be performed.Each cartridge will include a sensor array, which includes a pluralityof chemically sensitive particles, each of the particles includingreceptors specific for the desired test. For example, a sensor arraycartridge for use in medical testing for diabetes may include a numberof particles that are sensitive to sugars. A sensor array for use inwater testing, however, would include different particles, for example,particles specific for pH and/or metal ions.

The sensor array cartridge may be held in place in a manner analogous toa floppy disk of a computer. The sensor array cartridge may be inserteduntil it snaps into a holder disposed within the portable sensor system.The holder may inhibit the cartridge from falling out from the portablesensor system and place the sensor in an appropriate position to receivethe fluid samples. The holder may also align the sensor array cartridgewith the light source and the detector. A release mechanism may beincorporated into the holder that allows the cartridge to be releasedand ejected from the holder. Alternatively, the portable sensor arraysystem may incorporate a mechanical system for automatically receivingand ejecting the cartridge in a manner analogous to a CD-ROM typesystem.

The analysis of simple analyte species like acids/bases, salts, metals,anions, hydrocarbon fuels, and solvents may be repeated using highlyreversible receptors. Chemical testing of these species may berepeatedly accomplished with the same sensor array cartridge. In somecases, the cartridge may require a flush with a cleaning solution toremove traces from a previous test. Thus, replacement of cartridges forenvironmental usage may be required on an occasional basis (e.g., daily,weekly, or monthly) depending on the analyte and the frequency oftesting.

Alternatively, the sensor array may include highly specific receptors.Such receptors are particularly useful for medical testing, and testingfor chemical and biological warfare agents. Once a positive signal isrecorded with these sensor arrays, the sensor array cartridge may needto be replaced immediately. The use of a sensor array cartridge makesthis replacement easy.

Fluid samples may be introduced into the system at ports 1020 and 1022at the top of the unit. Two ports are shown, although more ports may bepresent. Port 1022 may be for the introduction of liquids found in theenvironment and some bodily fluids (e.g., water, saliva, urine, etc.).Port 1020 may be used for the delivery of human whole blood samples. Thedelivery of blood may be accomplished by the use of a pinprick to piercethe skin and a capillary tube to collect the blood sample. Port 1020 mayaccept either capillary tubes or syringes that include blood samples.

For the collection of environmental samples, syringe 1030 may be used tocollect the samples and transfer the samples to the input ports. Theportable sensor array system may include a holder that allows thesyringe to be coupled to the side of the portable sensor array system.Ports 1020 may include a standard Luer lock adapter (either male orfemale) to allow samples collected by syringe to be directly introducedinto the portable sensor array system from the syringe.

The input ports may also be used to introduce samples in a continuousmanner. The introduction of samples in a continuous manner may be used,e.g., to evaluate water streams. An external pump may be used tointroduce samples into the portable sensor array system in a continuousmanner. Alternatively, internal pumps disposed within the portablesensor array system may be activated to pull a continuous stream of thefluid sample into the portable sensor array system. The ports may allowintroduction of gaseous samples.

In some cases, it may be necessary to filter a sample prior to itsintroduction into the portable sensor array system. For example,environmental samples may be filtered to remove solid particles prior totheir introduction into the portable sensor array system. Commerciallyavailable nucleopore filters 1040 anchored at the top of the unit may beused for this purpose. In one embodiment, filters 1040 may have Luerlock connections (either male or female) on both sides allowing them tobe connected directly to an input port and a syringe.

In one embodiment, all of the necessary fluids required for thechemical/biochemical analyses are contained within the portable sensorarray system. The fluids may be stored in one or more cartridges 1050.Cartridges 1050 may be removable from the portable sensor array system.Thus, when cartridge 1050 is emptied of fluid, the cartridge may bereplaced by a new cartridge or removed and refilled with fluid.Cartridges 1050 may also be removed and replaced with cartridges filledwith different fluids when the sensor array cartridge is changed. Thus,the fluids may be customized for the specific tests being run. Fluidcartridges may be removable or may be formed as an integral part of thereader.

Fluid cartridges 1050 may include a variety of fluids for the analysisof samples. In one embodiment, each cartridge may include up to about 5mL of fluid and may deleted after about 100 tests. One or morecartridges 1050 may include a cleaning solution. The cleaning solutionmay be used to wash and/or recharge the sensor array prior to a newtest. In one embodiment, the cleaning solution may be a buffer solution.Another cartridge 1050 may include visualization agents.

Visualization agents may be used to create a detectable signal from theparticles of the sensor array after the particles interact with thefluid sample. In one embodiment, visualization agents include dyes(visible or fluorescent) or molecules coupled to a dye, which interactwith the particles to create a detectable signal. In an embodiment,cartridge 1050 may be a vacuum reservoir. The vacuum reservoir may beused to draw fluids into the sensor array cartridge. The vacuumcartridge would act in an analogous manner to the vacutainer cartridgesdescribed previously. In another embodiment, a fluid cartridge may beused to collect fluid samples after they pass through the sensor array.The collected fluid samples may be disposed of in an appropriate mannerafter the testing is completed.

In one embodiment, alphanumeric display screen 1014 may be used toprovide information relevant to the chemistry/biochemistry of theenvironment or blood samples. Also included within the portable sensorarray system may be a data communication system. Such systems includedata communication equipment for the transfer of numerical data, videodata, and/or sound data. Transfer may be accomplished using eitherdigital or analog standards. The data may be transmitted using anytransmission medium such as electrical wire, infrared, RF, and/or fiberoptic. In one embodiment, the data transfer system may include awireless link that may be used to transfer the digitalchemistry/biochemistry data to a closely positioned communicationspackage. In another embodiment, the data transfer system may include afloppy disk drive for recording the data and allowing the data to betransferred to a computer system. In another embodiment, the datatransfer system may include serial or parallel port connection hardwareto allow transfer of data to a computer system.

The portable sensor array system may also include a global positioningsystem (“GPS”). The GPS may be used to track the area from which asample is collected. After collecting sample data, the data may be fedto a server, which compiles the data along with GPS information.Subsequent analysis of this information may be used to generate achemical/biochemical profile of an area. For example, tests of standingwater sources in a large area may be used to determine the environmentaldistribution of pesticides or industrial pollutants.

Other devices may also be included in the portable sensor array that isspecific for other applications. For example, medical monitoring devicesmay include, but is not limited to, EKG monitors, blood pressuredevices, pulse monitors, and temperature monitors.

The detection system may be implemented in a number of different wayssuch that all of the detection components fit within the casing of theportable sensor array system. For an optical detection/imaging device,either CMOS or CCD focal plane arrays may be used. The CMOS detectoroffers some advantages in terms of lower cost and power consumption,while the CCD detector offers the highest possible sensitivity.Depending on the illumination system, either monochrome or colordetectors may be used. A one-to-one transfer lens may be employed toproject the image of a bead sensor array onto the focal plane of thedetector. All fluidic components may be sealed from contact with anyoptical or electronic components. Sealing the fluids from the detectorsavoids complications that may arise from contamination or corrosion insystems that require direct exposure of electronic components to thefluids under test. Other detectors such as photodiodes, cameras,integrated detectors, photoelectric cells, interferometers, andphotomultiplier tubes may be used.

The illumination system for colorimetric detection may be constructed inseveral manners. When using a monochrome focal plane array, amulti-color, but “discrete-wavelength-in-time” illumination system maybe used. The simplest implementation may include several LED's (lightemitting diodes) each operating at a different wavelength. Red, green,yellow, and blue wavelength LEDs is now commercially available for thispurpose. By switching from one LED to the next, and collecting an imageassociated with each, calorimetric data may be collected.

It is also possible to use a color focal plane detector array. A colorfocal plane detector may allow the determination of calorimetricinformation after signal acquisition using image processing methods. Inthis case, a “white light” illuminator is used as the light source.“White light” LEDs may be used as the light source for a color focalplane detector. White light LEDs use a blue LED coated with a phosphorto produce a broadband optical source. The emission spectrum of suchdevices may be suitable for calorimetric data acquisition. A pluralityof LEDs may be used. Alternatively, a single LED may be used.

Other light sources that may be useful include electroluminescentsources, fluorescent light sources, incandescent light sources, laserlights sources, laser diodes, arc lamps, and discharge lamps. The systemmay also use an external light source (both natural and unnatural) forillumination.

A lens may be positioned in front of the light source to allow theillumination area of the light source to be expanded. The lens may alsoallow the intensity of light reaching the sensor array to be controlled.For example, the illumination of the sensor array may be made uniform bythe use of a lens. In one example, a single LED light may be used toilluminate the sensor array. Examples of lenses that may be used inconjunction with an LED include Diffusing plate PN K43-717 Lens JML,PN61874 from Edmund scientific.

In addition to calorimetric signaling, chemical sensitizers may be usedthat produce a fluorescent response. The detection system may still beeither monochrome (for the case where the specific fluorescence spectrumis not of interest, just the presence of a fluorescence signal) orcolor-based (that would allow analysis of the actual fluorescencespectrum). An appropriate excitation notch filter (in one embodiment, along wavelength pass filter) may be placed in front of the detectorarray. The use of a fluorescent detection system may require anultraviolet light source. Short wavelength LEDs (e.g., blue to near UV)may be used as the illumination system for a fluorescent-based detectionsystem.

In some embodiments, use of a light source may not be necessary. Theparticles may rely on the use of chemiluminescence, thermoluminescenceor piezoluminescence to provide a signal. In the presence of an analyteof interest, the particle may be activated such that the particlesproduce light. In the absence of an analyte, the particles may produceminimal or no light.

The portable sensor array system may also include an electroniccontroller, which controls the operation of the portable sensor arraysystem. The electronic controller may also be capable of analyzing thedata and determining the identity of the analytes present in a sample.While the electronic controller is described herein for use with theportable sensor array system, it should be understood that theelectronic controller might be used with any of the previously describedembodiments of an analyte detection system.

The controller may be used to control the various operations of theportable sensor array. Some of the operations that may be controlled ormeasured by the controller include: (i) determining the type of sensorarray present in the portable sensor array system; (ii) determining thetype of light required for the analysis based on the sensor array; (iii)determining the type of fluids required for the analysis, based on thesensor array present; (iv) collecting the data produced during theanalysis of the fluid sample; (v) analyzing the data produced during theanalysis of the fluid sample; (vi) producing a list of the componentspresent in the inputted fluid sample; and, (vii) monitoring samplingconditions (e.g., temperature, time, density of fluid, turbidityanalysis, lipemia, bilirubinemia, etc).

Additionally, the controller may provide system diagnostics andinformation to the operator of the apparatus. The controller may notifythe user when routine maintenance is due or when a system error isdetected. The controller may also manage an interlock system for safetyand energy conservation purposes. For example, the controller mayprevent the lamps from operating when the sensor array cartridge is notpresent.

The controller may also interact with an operator. The controller mayinclude input device 1012 and display screen 1014, as depicted in FIG.18. A number of operations controlled by the controller, as describedabove, may be dependent on the input of the operator. The controller mayprepare a sequence of instructions based on the type of analysis to beperformed. The controller may send messages to the output screen to letthe used know when to introduce samples for the test and when theanalysis is complete. The controller may display the results of anyanalysis performed on the collected data on the output screen.

Many of the testing parameters may be dependent upon the type of sensorarray used and the type of sample being collected. The controller willrequire, in some embodiments, the identity of the sensor array and testbeing performed in order to set up the appropriate analysis conditions.Information concerning the sample and the sensor array may be collectedin a number of manners.

In one embodiment, the sample and sensor array data may be directlyinputted by the user to the controller. Alternatively, the portablesensor array may include a reading device, which determines the type ofsensor cartridge being used once the cartridge is inserted. In oneembodiment, the reading device may be a bar code reader capable ofreading a bar code placed on the sensor array. In this manner, thecontroller can determine the identity of the sensor array without anyinput from the user. In another embodiment, the reading device may bemechanical in nature. Protrusions or indentation formed on the surfaceof the sensor array cartridge may act as a code for a mechanical readingdevice. The information collected by the mechanical reading device maybe used to identify the sensor array cartridge. Other devices may beused to accomplish the same function as the bar code reader. Thesedevices include smart card readers and RFID systems.

The controller may also accept information from the user regarding thetype of test being performed. The controller may compare the type oftest being performed with the type of sensor array present in theportable sensor array system. If an inappropriate sensor array cartridgeis present, an error message may be displayed and the portable sensorarray system may be disabled until the proper cartridge is inserted. Inthis manner, incorrect testing resulting from the use of the wrongsensor cartridge may be avoided.

The controller may also monitor the sensor array cartridge and determineif the sensor array cartridge is functioning properly. The controllermay run a quick analysis of the sensor array to determine if the sensorarray has been used and if any analytes are still present on the sensorarray. If analytes are detected, the controller may initiate a cleaningsequence, where a cleaning solution is passed over the sensor arrayuntil no more analytes are detected. Alternatively, the controller maysignal the user to replace the cartridge before testing is initiated.

Another embodiment of a portable sensor array system is depicted inFIGS. 19A-B. In this embodiment, portable sensor array 1100 includesbody 1110 that holds the various components used with the sensor arraysystem. A sensor array, such as the sensor arrays described herein, maybe placed in cartridge 1120. Cartridge 1120 may support the sensor arrayand allow the proper positioning of the sensor array within the portablesensor system.

A schematic cross-sectional view of the body of the portable sensorarray system is depicted in FIG. 19B. Cartridge 1120, in which thesensor array is disposed, extends into body 1110. Within the body, lightsource 1130 and detector 1140 are positioned proximate to cartridge1120. When cartridge 1120 is inserted into the reader, the cartridge maybe held by body 110 at a position proximate to the location of thesensor array within the cartridge. Light source 1130 and detector 1140may be used to analyze samples disposed within the cartridge. Electroniccontroller 1150 may be coupled to detector 1140. Electronic controller1150 may be used to receive data collected by the portable sensor arraysystem. The electronic controller may also be used to transmit datacollected to a computer.

An embodiment of a cartridge for use in a sensor array system isdepicted in FIG. 20. Cartridge 1200 includes carrier body 1210 that isformed of a material that is substantially transparent to a wavelengthof light used by the detector. In an embodiment, plastic materials maybe used. Examples of plastic materials that may be used includepolycarbonates and polyacrylates. In one embodiment, body 1210 may beformed from a Cyrolon AR2 Abrasion Resistant polycarbonate sheet at athickness of about 0.118 inches and about 0.236 inches. Sensor arraygasket 1220 may be placed on carrier body 1210. Sensor array gasket 1220may help reduce or inhibit the amount of fluids leaking from the sensorarray. Leaking fluids may interfere with the testing being performed.

Sensor array 1230 may be placed onto sensor array gasket 1220. Thesensor array may include one or more cavities, each of which includesone or more particles disposed within the cavities. The particles mayreact with an analyte present in a fluid to produce a detectable signal.Any of the sensor arrays described herein may be used in conjunctionwith the portable reader.

Second gasket 1240 may be positioned on sensor array 1230. Second gasket1240 may be disposed between sensor array 1230 and window 1250. Secondgasket 1240 may form a seal inhibiting leakage of the fluid from thesensor array. Window 1250 may be disposed above the gasket to inhibitdamage to the sensor array.

Coupling cover 1270 to body 1210 may complete the assembly. Rubbergasket 1260 may be disposed between the cover and the window to reducepressure exerted by the cover on the window. The cover may seal thesensor array, gaskets, and window into the cartridge. The sensor array,gaskets and window may all be sealed together using a pressure sensitiveadhesive. An example of a pressure sensitive adhesive is Optimount 237made by Seal products. Gaskets may be made from polymeric materials. Inone example, Calon II—High Performance material from Arlon may be used.The rubber spring may be made from a silicon rubber material.

The cover may be removable or sealed. When a removable cover is used,the cartridge may be reused by removing the cover and replacing thesensor array. Alternatively, the cartridge may be a one-use cartridge inwhich the sensor array is sealed within the cartridge.

The cartridge may also include reservoir 1280. The reservoir may hold ananalyte containing fluid after the fluids pass through the sensor array.FIG. 21 depicts a cut away view of the cartridge that shows thepositions of channels formed in the cartridge. The channels may allowthe fluids to be introduced into the cartridge. The channels also mayconduct the fluids from the inlet to the sensor array and to thereservoir.

In one embodiment, cartridge body 1210 includes a number of channelsdisposed throughout the body. Inlet port 1282 may receive a fluiddelivery device for the introduction of fluid samples into thecartridge. In one embodiment, the inlet port may include a Luer lockadapter to couple with a corresponding Luer lock adapter on the fluiddelivery device. For example, a syringe may be used as the fluiddelivery device. The Luer lock fitting on the syringe may be coupledwith a mating Luer lock fitting on inlet port 1282. Luer lock adaptersmay also be coupled to tubing, so that fluid delivery may beaccomplished by the introduction of fluids through appropriate tubing tothe cartridge.

Fluid passes through channel 1284 to channel outlet 1285. Channel outlet1285 may be coupled to an inlet port on a sensor array. Channel outlet1285 is also depicted in FIG. 20. The fluid travels into the sensorarray and through the cavities. After passing through the cavities, thefluid exits the sensor array and enters channel 1286 via channel inlet1287. The fluid passes through channel 1286 to reservoir 1280. Tofacilitate the transfer of fluids through the cartridge, the reservoirmay include air outlet port 1288. Air outlet port 1288 may allow air topass out of the reservoir, while retaining any fluids disposed withinthe reservoir. In one embodiment, air outlet port 1288 may be an openingformed in the reservoir that is covered by a semipermeable membrane. Acommercially available air outlet port includes a DURAVENT containervent, available from W. L. Gore. It should be understood, however, thatany other material that allows air to pass out of the reservoir, whileretaining fluids in the reservoir, might be used. After extended use,reservoir 1280 may become filled with fluids. Outlet channel 1290 mayalso be formed extending through body 1210 to allow removal of fluidsfrom the body. Fluid cartridges 1292 for introducing additional fluidsinto the sensor array may be incorporated into the cartridges.

Herein we describe a system and method for the collection andtransmission of chemical information over a computer network. Thesystem, in some embodiments, includes an analyte detection device(“ADD”) operable to detect one or more analytes or mixtures of analytesin a fluid containing one or more analytes, and computer hardware andsoftware operable to send and receive data over a computer network toand from a client computer system.

Chemical information refers to any data representing the detection of aspecific chemical or a combination of chemicals. These data may include,but are not limited to chemical identification, chemical proportions, orvarious other forms of information related to chemical detection. Theinformation may be in the form of raw data, including binary oralphanumeric, formatted data, or reports. In some embodiments, chemicalinformation relates to data collected from an analyte detection device.Such data includes data related to the color of the particles includedon the analyte detection device. The chemical information collected fromthe analyte detection device may include raw data (e.g., a color, RBGdata, intensity at a specific wavelength) etc. Alternatively, the datamay be analyzed by the analyte detection device to determine theanalytes present. The chemical information may include the identities ofthe analytes detected in the fluid sample. The information may beencrypted for security purposes.

In one embodiment, the chemical information may be in LogicalObservation Identifiers Names and Codes (LOINC) format. The LOINC formatprovides a standard set of universal names and codes for identifyingindividual laboratory results (e.g. hemoglobin, serum sodiumconcentration), clinical observations (e.g. discharge diagnosis,diastolic blood pressure) and diagnostic study observations, (e.g.PR-interval, cardiac echo left ventricular diameter, chest x-rayimpression).

More specifically, chemical information may take the form of datacollected by the analyte detection system. As described above, ananalyte detection system may include a sensor array that includes aparticle or particles. These particles may produce a detectable signalin response to the presence or absence of an analyte. The signal may bedetected using a detector. The detector may detect the signal. Thedetector may also produce an output signal that contains informationrelating to the detected signal. The output signal may, in someembodiments be the chemical information.

In some embodiments, the detector may be a light detector and the signalproduced by the particles may be modulated light. The detector mayproduce an output signal that is representative of the detected lightmodulation. The output signal may be representative of the wavelength ofthe light signal detected. Alternatively, the output signal may berepresentative of the strength of the light signal detected. In otherembodiments, the output signal may include both wavelength and strengthof signal information.

In some embodiments, use of a light source may not be necessary. Theparticles may rely on the use of chemiluminescence, thermoluminescenceor piezoluminescence to provide a signal. In the presence of an analyteof interest, the particle may be activated such that the particlesproduce light. In the absence of an analyte, the particles may notexhibit produce minimal or no light. The chemical information may berelated to the detection or absence of a light produced by theparticles, rather than modulated by the particles.

The detector output signal information may be analyzed by analysissoftware. The analysis software may convert the raw output data tochemical information that is representative of the analytes in theanalyzed fluid system. The chemical information may be either the rawdata before analysis by the computer software or the informationgenerated by processing of the raw data.

The term “computer system” as used herein generally describes thehardware and software components that in combination allow the executionof computer programs. The computer programs may be implemented insoftware, hardware, or a combination of software and hardware. Computersystem hardware generally includes a processor, memory media, andinput/output (I/O) devices. As used herein, the term “processor”generally describes the logic circuitry that responds to and processesthe basic instructions that operate a computer system. The term “memorymedium” includes an installation medium, e.g., a CD-ROM, floppy disks; avolatile computer system memory such as DRAM, SRAM, EDO RAM, Rambus RAM,etc.; or a non-volatile memory such as optical storage or a magneticmedium, e.g., a hard drive. The term “memory” is used synonymously with“memory medium” herein. The memory medium may comprise other types ofmemory or combinations thereof. In addition, the memory medium may belocated in a first computer in which the programs are executed, or maybe located in a second computer that connects to the first computer overa network. In the latter instance, the second computer provides theprogram instructions to the first computer for execution. In addition,the computer system may take various forms, including a personalcomputer system, mainframe computer system, workstation, networkappliance, Internet appliance, personal digital assistant (PDA),television system or other device. In general, the term “computersystem” can be broadly defined to encompass any device having aprocessor that executes instructions from a memory medium.

The memory medium may stores a software program or programs for thereception, storage, analysis, and transmittal of information produced byan Analyte Detection Device (ADD). The software program(s) may beimplemented in any of various ways, including procedure-basedtechniques, component-based techniques, and/or object-orientedtechniques, among others. For example, the software program may beimplemented using ActiveX controls, C++ objects, JavaBeans, MicrosoftFoundation Classes (MFC), or other technologies or methodologies, asdesired. A central processing unit (CPU), such as the host CPU, forexecuting code and data from the memory medium includes a means forcreating and executing the software program or programs according to themethods, flowcharts, and/or block diagrams described below.

A computer system's software generally includes at least one operatingsystem such as Windows NT, Windows 95, Windows 98, or Windows ME (allavailable from Microsoft Corporation); Mac OS and Mac OS X Server (AppleComputer, Inc.), MacNFS (Thursby Software), PC MACLAN (Miramar Systems),or real time operating systems such as VXWorks (Wind River Systems,Inc.), QNX (QNX Software Systems, Ltd.), etc. The foregoing are allexamples of specialized software programs that manage and provideservices to other software programs on the computer system. Software mayalso include one or more programs to perform various tasks on thecomputer system and various forms of data to be used by the operatingsystem or other programs on the computer system. Software may also beoperable to perform the functions of an operating system (OS). The datamay include but is not limited to databases, text files, and graphicsfiles. A computer system's software generally is stored in non-volatilememory or on an installation medium. A program may be copied into avolatile memory when running on the computer system. Data may be readinto volatile memory as the data is required by a program.

A server program may be defined as a computer program that, whenexecuted, provides services to other computer programs executing in thesame or other computer systems. The computer system on which a serverprogram is executing may be referred to as a server, though it maycontain a number of server and client programs. In the client/servermodel, a server program awaits and fulfills requests from clientprograms in the same or other computer systems. Examples of computerprograms that may serve as servers include: Windows NT (MicrosoftCorporation), Mac OS X Server (Apple Computer, Inc.), MacNFS (ThursbySoftware), PC MACLAN (Miramar Systems), etc

A web server is a computer system, which maintains a web site browsableby any of various web browser software programs. As used herein, theterm ‘web browser’ refers to any software program operable to access websites over a computer network.

An intranet is a network of networks that is contained within anenterprise. An intranet may include many interlinked local area networks(LANs) and may use data connections to connect LANs in a wide areanetwork (WAN). An intranet may also include connections to the Internet.An intranet may use TCP/IP, HTTP, and other Internet protocols.

An extranet, or virtual private network, is a private network that usesInternet protocols and public telecommunication systems to securelyshare part of a business' information or operations with suppliers,vendors, partners, customers, or other businesses. An extranet may beviewed as part of a company's intranet that is extended to users outsidethe company. An extranet may require security and privacy. Companies mayuse an extranet to exchange large volumes of data, share productcatalogs exclusively with customers, collaborate with other companies onjoint development efforts, provide or access services provided by onecompany to a group of other companies, and to share news of commoninterest exclusively with partner companies.

Connection mechanisms included in a network may include copper lines,optical fiber, radio transmission, satellite relays, or any other deviceor mechanism operable to allow computer systems to communicate.

As used herein, ADD refers to any device or instrument operable todetect one or more specific analytes or mixtures of analytes in a fluidsample, wherein the fluid sample may be liquid, gaseous, solid, asuspension of a solid in a gas, or a suspension of a liquid in a gas.More particularly, an ADD includes a sensor array, light and detectorare described in U.S. Patent Application Publication No. US 2002-0197622A1, which is fully incorporated herein by reference as if set forthherein.

A particle, in some embodiments, possesses both the ability to bind theanalyte of interest and to create a modulated signal. The particle mayinclude receptor molecules which posses the ability to bind the analyteof interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte of interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner. The forces, which driveassociation/recognition between molecules, include the hydrophobiceffect, anion-cation attraction, and hydrogen bonding. The relativestrengths of these forces depend upon factors such as the solventdielectric properties, the shape of the host molecule, and how itcomplements the guest. Upon host-guest association, attractiveinteractions occur and the molecules stick together. The most widelyused analogy for this chemical interaction is that of a “lock and key”.The fit of the key molecule (the guest) into the lock (the host) is amolecular recognition event.

A naturally occurring or synthetic receptor may be bound to a polymericresin in order to create the particle. The polymeric resin may be madefrom a variety of polymers including, but not limited to, agarous,dextrose, acrylamide, control pore glass beads, polystyrene-polyethyleneglycol resin, polystyrene-divinyl benzene resin, formylpolystyreneresin, trityl-polystyrene resin, acetyl polystyrene resin, chloroacetylpolystyrene resin, aminomethyl polystyrene-divinylbenzene resin,carboxypolystyrene resin, chloromethylated polystyrene-divinylbenzeneresin, hydroxymethyl polystyrene-divinylbenzene resin, 2-chlorotritylchloride polystyrene resin, 4-benzyloxy-2′4′-dimethoxybenzhydrol resin(Rink Acid resin), triphenyl methanol polystyrene resin,diphenylmethanol resin, benzhydrol resin, succinimidyl carbonate resin,p-nitrophenyl carbonate resin, imidazole carbonate resin, polyacrylamideresin, 4-sulfamylbenzoyl-4′- methylbenzhydrylamine-resin (Safety-catchresin), 2-amino-2-(2′-nitrophenyl)propionic acid-aminomethyl resin (ANPResin), p-benzyloxybenzyl alcohol-divinylbenzene resin (Wang resin),p-methylbenzhydrylamine-divinylbenzene resin (MBHA resin),Fmoc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine linked to resin(Knorr resin), 4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin(Rink resin), 4-hydroxymethyl-benzoyl-4′- methylbenzhydrylamine resin(HMBA-MBHA Resin), p-nitrobenzophenone oxime resin (Kaiser oxime resin),and amino-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine handlelinked to 2-chlorotrityl resin (Knorr-2-chlorotrityl resin). In oneembodiment, the material used to form the polymeric resin is compatiblewith the solvent in which the analyte is dissolved. For example,polystyrene-divinyl benzene resin will swell within non-polar solvents,but does not significantly swell within polar solvents. Thus,polystyrene-divinyl benzene resin may be used for the analysis ofanalytes within non-polar solvents. Alternatively,polystyrene-polyethylene glycol resin will swell with polar solventssuch as water. Polystyrene-polyethylene glycol resin may be useful forthe analysis of aqueous fluids.

In one embodiment, a polystyrene-polyethylene glycol-divinyl benzenematerial is used to form the polymeric resin. Thepolystyrene-polyethylene glycol-divinyl benzene resin is formed from amixture of polystyrene 1400, divinyl benzene 1420 andpolystyrene-polyethylene glycol 1440 (see FIG. 22). The polyethyleneglycol portion of the polystyrene-polyethylene glycol 1440, in oneembodiment, may be terminated with an amine. The amine serves as achemical handle to anchor both receptors and indicator dyes. Otherchemical functional groups may be positioned at the terminal end of thepolyethylene glycol to allow appropriate coupling of the polymeric resinto the receptor molecules or indicators.

The chemically sensitive particle, in one embodiment, is capable of bothbinding the analyte(s) of interest and creating a detectable signal. Inone embodiment, the particle will create an optical signal when bound toan analyte of interest. The use of such a polymeric bound receptorsoffers advantages both in terms of cost and configurability. Instead ofhaving to synthesize or attach a receptor directly to a supportingmember, the polymeric bound receptors may be synthesized en masse anddistributed to multiple different supporting members. This allows thecost of the sensor array, a major hurdle to the development ofmass-produced environmental probes and medical diagnostics, to bereduced. Additionally, sensor arrays, which incorporate polymeric boundreceptors, may be reconfigured much more quickly than array systems inwhich the receptor is attached directly to the supporting member. Forexample, if a new variant of a pathogen or a pathogen that contains agenetically engineered protein is a threat, then a new sensor arraysystem may be readily created to detect these modified analytes bysimply adding new sensor elements (e.g., polymeric bound receptors) to apreviously formed supporting member.

Systems in which receptors are sensitive to changes in pH are describedin U.S. patent application Ser. No. 09/287,248; U.S. Pat. Nos.6,680,206; 6,649,403; and 6,713,298; and U.S. Patent ApplicationPublication Nos. US 2003-0064422 A1; US 2004-0053322 A1; US 2003-0186228A1; and US 2002-0197622 A1, which incorporated herein by reference as ifset forth herein. In these systems, a receptor, which is sensitive tochanges in the pH of a fluid sample, is bound to a polymeric resin tocreate a particle. That is, the receptor is sensitive to theconcentration of hydrogen cations (H⁺). The receptor in this case istypically sensitive to the concentration of H⁺ in a fluid solution. Theanalyte of interest may therefore be H⁺. There are many types ofmolecules, which undergo a color change when the pH of the fluid ischanged.

Systems in which receptors are sensitive to the concentrations of one ormore metal cations present in a fluid solution are described in U.S.patent application Ser. No. 09/287,248; U.S. Pat. Nos. 6,680,206;6,649,403; and 6,713,298; and U.S. Patent Application Publication Nos.US 2003-0064422 A1; US 2004-0053322 A1; US 2003-0186228 A1; and US2002-0197622 A1, which are incorporated herein by reference as if setforth herein. In these systems, the receptor in this case is typicallysensitive to the concentration of one or more metal cations present in afluid solution. In general, colored molecules, which will bind cations,may be used to determine the presence of a metal cation in a fluidsolution.

In one embodiment, a detectable signal may be caused by the altering ofthe physical properties of an indicator ligand bound to the receptor orthe polymeric resin. In one embodiment, two different indicators areattached to a receptor or the polymeric resin. When an analyte iscaptured by the receptor, the physical distance between the twoindicators may be altered such that a change in the spectroscopicproperties of the indicators is produced. A variety of fluorescent andphosphorescent indicators may be used for this sensing scheme. Thisprocess, known as Forster energy transfer, is extremely sensitive tosmall changes in the distance between the indicator molecules.

For example, first fluorescent indicator 1460 (e.g., a fluoresceinderivative) and second fluorescent indictor 1480 (e.g., a rhodaminederivative) may be attached to receptor 1500, as depicted in FIG. 23.When no analyte is present, short wavelength excitation 1520 may excitefirst fluorescent indicator 1460, which fluoresces as indicated by 1540.The short wavelength excitation, however, may cause little or nofluorescence of second fluorescent indicator 1480. After binding ofanalyte 1560 to the receptor, a structural change in the receptormolecule may bring the first and second fluorescent indicators closer toeach other. This change in intermolecular distance may allow an excitedfirst indicator 1460 to transfer a portion of fluorescent energy 1580 tosecond fluorescent indicator 1480. This transfer in energy may bemeasured by either a drop in energy of the fluorescence of firstindicator molecule 1460, or the detection of increased fluorescence 1600by second indicator molecule 1480.

Alternatively, first and second fluorescent indicators 1460 and 1480,respectively, may initially be positioned such that short wavelengthexcitation causes fluorescence of both the first and second fluorescentindicators, as described above. After binding of analyte 1560 to thereceptor, a structural change in the receptor molecule may cause thefirst and second fluorescent indicators to move, further apart. Thischange in intermolecular distance may inhibit the transfer offluorescent energy from first indicator 1460 to second fluorescentindicator 1480. This change in the transfer of energy may be measured byeither a drop in energy of the fluorescence of second indicator molecule1480, or the detection of increased fluorescence by first indicatormolecule 1460.

In another embodiment, an indicator ligand may be preloaded onto thereceptor. An analyte may then displace the indicator ligand to produce achange in the spectroscopic properties of the particles. In this case,the initial background absorbance is relatively large and decreases whenthe analyte is present. The indicator ligand, in one embodiment, has avariety of spectroscopic properties, which may be measured. Thesespectroscopic properties include, but are not limited to, ultravioletabsorption, visible absorption, infrared absorption, fluorescence, andmagnetic resonance. In one embodiment, the indicator is a dye having astrong fluorescence, a strong ultraviolet absorption, a strong visibleabsorption, or a combination of these physical properties. Examples ofindicators include, but are not limited to, carboxyfluorescein, ethidiumbromide, 7-dimethylamino-4-methylcoumarin,7-diethylamino-4-methylcoumarin, eosin, erythrosin, fluorescein, OregonGreen 488, pyrene, Rhodamine Red, tetramethylrhodamine, Texas Red,Methyl Violet, Crystal Violet, Ethyl Violet, Malachite green, MethylGreen, Alizarin Red S, Methyl Red, Neutral Red,o-cresolsulfonephthalein, o-cresolphthalein, phenolphthalein, AcridineOrange, B-naphthol, coumarin, and a-naphthionic acid.

When the indicator is mixed with the receptor, the receptor andindicator interact with each other such that the above-mentionedspectroscopic properties of the indicator, as well as otherspectroscopic properties, may be altered. The nature of this interactionmay be a binding interaction, wherein the indicator and receptor areattracted to each other with a sufficient force to allow the newlyformed receptor-indicator complex to function as a single unit. Thebinding of the indicator and receptor to each other may take the form ofa covalent bond, an ionic bond, a hydrogen bond, a van der Waalsinteraction, or a combination of these bonds.

The indicator may be chosen such that the binding strength of theindicator to the receptor is less than the binding strength of theanalyte to the receptor. Thus, in the presence of an analyte, thebinding of the indicator with the receptor may be disrupted, releasingthe indicator from the receptor. When released, the physical propertiesof the indicator may be altered from those it exhibited when bound tothe receptor. The indicator may revert to its original structure, thusregaining its original physical properties. For example, if afluorescent indicator is attached to a particle that includes areceptor, the fluorescence of the particle may be strong beforetreatment with an analyte-containing fluid. When the analyte interactswith the particle, the fluorescent indicator may be released. Release ofthe indicator may cause a decrease in the fluorescence of the particle,since the particle now has less indicator molecules associated with it.

In another embodiment, a designed synthetic receptor may be used. In oneembodiment, a polycarboxylic acid receptor may be attached to apolymeric resin. The polycarboxylic receptors are discussed in U.S. Pat.No. 6,045,579, which is incorporated herein by reference.

In an embodiment, the analyte molecules in the fluid may be pretreatedwith an indicator ligand. Pretreatment may involve covalent attachmentof an indicator ligand to the analyte molecule. After the indicator hasbeen attached to the analyte, the fluid may be passed over the sensingparticles. Interaction of the receptors on the sensing particles withthe analytes may remove the analytes from the solution. Since theanalytes include an indicator, the spectroscopic properties of theindicator may be passed onto the particle. By analyzing the physicalproperties of the sensing particles after passage of an analyte stream,the presence and concentration of an analyte may be determined.

For example, the analytes within a fluid may be derivatized with afluorescent tag before introducing the stream to the particles. Asanalyte molecules are adsorbed by the particles, the fluorescence of theparticles may increase. The presence of a fluorescent signal may be usedto determine the presence of a specific analyte. Additionally, thestrength of the fluorescence may be used to determine the amount ofanalyte within the stream.

In one embodiment, a chromogenic signal generating process may beperformed to produce a color change on a particle. An analyte fluidintroduced into the cavity and reacted with the receptor. After thereaction period, an indicator may be added to the cavity. Theinteraction of the indicator with the receptor-analyte may produce adetectable signal. A particle, which has not been exposed to the analytemay remain unchanged or show a different color change. In an embodiment,a staining or precipitation technique may be used to further visualizethe indicator molecule. After a receptor-analyte-indicator complex isformed, a fluid containing a molecule that will react with the indicatorportion of the complex may be added to the cavity to cause a signalchange of the complex. A particle, which has not been exposed to theanalyte may remain unchanged or show a different color change.Optionally, a wash to remove unbound indicator molecules may beperformed before visualization of the receptor-analyte-indicatorcomplex. Examples of indicators may be, but are not limited to,fluorescent dyes, enzyme-linked molecules and/or colloidal preciousmetal linked molecules.

The development of smart sensors capable of discriminating differentanalytes, toxins, and/or bacteria has become increasingly important forenvironmental, health and safety, remote sensing, military, and chemicalprocessing applications. Although many sensors capable of highsensitivity and high selectivity detection have been fashioned forsingle analyte detection, only in a few selected cases have arraysensors been prepared which display multi-analyte detectioncapabilities. The obvious advantages of such array systems are theirutility for the analysis of multiple analytes and their ability to be“trained” to respond to new stimuli. Such on site adaptive analysiscapabilities afforded by the array structures may make their utilizationpromising for a variety of future applications.

Single and multiple analyte sensors typically rely on changes in opticalsignals. These sensors may make use of an indicator that undergoes aperturbation upon analyte binding. The indicator may be a chromophore ora fluorophore. A fluorophore is a molecule that absorbs light at acharacteristic wavelength and then re-emits the light at acharacteristically different wavelength. Fluorophores include, but arenot limited to, rhodamine and rhodamine derivatives, fluorescein andfluorescein derivatives, coumarins, and chelators with the lanthanideion series. The emission spectra, absorption spectra, and chemicalcomposition of many fluorophores may be found, e.g., in the “Handbook ofFluorescent Probes and Research Chemicals”, R. P. Haugland, ed. which isincorporated herein by reference. A chromophore is a molecule whichabsorbs light at a characteristic wavelength, but does not re-emitlight.

As previously described, the receptor itself may incorporate anindicator. The binding of the analyte to the receptor may directly leadto a modulation of the properties of the indicator. Such an approachtypically requires a covalent attachment or strong non-covalent bindingof the indicator onto or as part of the receptor, leading to additionalcovalent architecture. Every receptor may need a designed signalingprotocol that is typically unique to that receptor. General protocolsfor designing signal modulation that is versatile for most any receptorwould be desirable.

In one embodiment, a general method for the creation of optical signalmodulations for most any receptor coupled to an immobilized matrix isdeveloped. Immobilized matrices include, but are not limited to, resins,beads, and polymer surfaces. By immobilization of the receptor to thematrix, the receptor is held within a structure that can be chemicallymodified, allowing one to tune and to create an environment around thereceptor that is sensitive to analyte binding. Coupling of the indicatorto an immobilization matrix may make it sensitive to microenvironmentchanges, which foster signal modulation of the indicator upon analytebinding. Further, by coupling the indicator to an immobilization matrix,the matrix itself becomes the signaling unit, not requiring a specificnew signaling protocol for every receptor immobilized on the matrix.

In an embodiment, a receptor for a particular analyte or class ofanalytes may be designed and created with the chemical handlesappropriate for immobilization on and/or in the matrix. A number of suchreceptors have been described above. The receptors can be, but are notlimited to, antibodies, aptamers, organic receptors, combinatoriallibraries, enzymes, and imprinted polymers.

Signaling indicator molecules may be created or purchased which haveappropriate chemical handles for immobilization on and/or in theimmobilization matrix. The indicators may possess chromophores orfluorophores that are sensitive to their microenvironment. Thischromophore or fluorophore may be sensitive to microenvironment changesthat include, but are not limited to, sensitivity to local pH,solvatophobic or solvatophilic properties, ionic strength, dielectric,ion pairing, and/or hydrogen bonding. Common indicators, dyes, quantumparticles, and semi-conductor particles, are all examples of possibleprobe molecules. The probe molecules may have epitopes similar to theanalyte, so that a strong or weak association of the probe moleculeswith the receptor may occur. Alternatively, the probe molecules may besensitive to a change in their microenvironment that results from one ofthe affects listed in item above.

Binding of the analyte may do one of the following things, resulting ina signal modulation: 1) displace a probe molecule from the binding siteof the receptor, 2) alter the local pH, 3) change the local dielectricproperties, 4) alter the features of the solvent, 5) change thefluorescence quantum yield of individual dyes, 6) alter therate/efficiency of fluorescence resonance energy transfer (FRET) betweendonor-acceptor fluorophore pairs, or 7) change the hydrogen bonding orion pairing near the probe.

In an alternative embodiment, two or more indicators may be attached tothe matrix. Binding between the receptor and analyte causes a change inthe communication between the indicators, again via either displacementof one or more indicators, or changes in the microenvironment around oneor more indicators. The communication between the indicators may be, butis not limited to, fluorescence resonance energy transfer, quenchingphenomenon, and/or direct binding.

In an embodiment, a particle for detecting an analyte may be composed ofa polymeric resin. A receptor and an indicator may be coupled to thepolymeric resin. The indicator and the receptor may be positioned on thepolymeric resin such that the indicator produces a signal in when theanalyte interacts with the receptor. The signal may be a change inabsorbance (for chromophoric indicators) or a change in fluorescence(for fluorophoric indicators).

A variety of receptors may be used in one embodiment; the receptor maybe a polynucleotide, a peptide, an oligosaccharide, an enzyme, a peptidemimetic, or a synthetic receptor. These receptors are described in U.S.Patent Application Publication No. US 2002-0197622 A1, which isincorporated by reference as if fully set forth herein.

A number of combinations for the coupling of an indicator and a receptorto a polymeric resin have been devised. These combinations areschematically depicted in FIGS. 24A-I. In one embodiment, depicted inFIG. 24A, receptor R may be coupled to a polymeric resin. The receptormay be directly formed on the polymeric resin, or be coupled to thepolymeric resin via a linker. Indicator I may also be coupled to thepolymeric resin. The indicator may be directly coupled to the polymericresin or coupled to the polymeric resin by a linker. In someembodiments, the linker coupling the indicator to the polymeric resin isof sufficient length to allow the indicator to interact with thereceptor in the absence of analyte A.

In another embodiment, depicted in FIG. 24B, receptor R may be coupledto a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker. Anindicator B may also be coupled to the polymeric resin. The indicatormay be directly coupled to the polymeric resin or coupled to thepolymeric resin by a linker. In some embodiments, the linker couplingthe indicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of analyte A.

An additional indicator C may also be coupled to the polymeric resin.The additional indicator may be directly coupled to the polymeric resinor coupled to the polymeric resin by a linker. In some embodiments, theadditional indicator is coupled to the polymeric resin, such that theadditional indicator is proximate the receptor during use.

In another embodiment, depicted in FIG. 24C, receptor R may be coupledto a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker.Indicator I may be coupled to the receptor. The indicator may bedirectly coupled to the receptor or coupled to the receptor by a linker.In some embodiments, the linker coupling the indicator to the polymericresin is of sufficient length to allow the indicator to interact withthe receptor in the absence of analyte A, as depicted in FIG. 24E.

In another embodiment, depicted in FIG. 24D, receptor R may be coupledto a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker.Indicator B may be coupled to the receptor. Indicator B may be directlycoupled to the receptor or coupled to the receptor by a linker. In someembodiments, the linker coupling the indicator to the polymeric resin isof sufficient length to allow the indicator to interact with thereceptor in the absence of analyte A. An additional indicator C may alsobe coupled to the receptor. The additional indicator may be directlycoupled to the receptor or coupled to the receptor by a linker asdepicted in FIG. 24F.

In another embodiment, depicted in FIG. 24G, receptor R may be coupledto a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker.Indicator B may be coupled to the polymeric resin. The indicator may bedirectly coupled to the polymeric resin or coupled to the polymericresin by a linker. In some embodiments, the linker coupling theindicator to the polymeric resin is of sufficient length to allow theindicator to interact with the receptor in the absence of analyte A. Anadditional indicator C may also be coupled to the receptor. Theadditional indicator may be directly coupled to the receptor or coupledto the receptor by a linker.

In another embodiment, depicted in FIG. 24H, receptor R may be coupledto a polymeric resin by a first linker. Indicator I may be coupled tothe first linker. The indicator may be directly coupled to the firstlinker or coupled to the first linker by a second linker. In someembodiments, the second linker coupling the indicator to the polymericresin is of sufficient length to allow the indicator to interact withthe receptor in the absence of analyte A.

In another embodiment, depicted in FIG. 24I, a receptor R may be coupledto a polymeric resin by a first linker. An indicator B may be coupled tothe first linker. The indicator may be directly coupled to the firstlinker or coupled to the first linker by a second linker. In someembodiments, the second linker coupling the indicator to the firstlinker is of sufficient length to allow the indicator to interact withthe receptor in the absence of analyte A. An additional indicator C maybe coupled to the receptor. The additional indicator may be directlycoupled to the receptor or coupled to the receptor by a linker.

These various combinations of receptors, indicators, linkers andpolymeric resins may be used in a variety of different signalingprotocols. Analyte-receptor interactions may be transduced into signalsthrough one of several mechanisms. In one approach, the receptor sitemay be preloaded with an indicator, which can be displaced in acompetition with analyte ligand. In this case, the resultant signal isobserved as a decrease in a signal produced by the indicator. Thisindicator may be a fluorophore or a chromophore. In the case of afluorophore indicator, the presence of an analyte may be determined by adecrease in the fluorescence of the particle. In the case of achromophore indicator, the presence of an analyte may be determined by adecrease in the absorbance of the particle.

A second approach that has the potential to provide better sensitivityand response kinetics is the use of an indicator as a monomer in thecombinatorial sequences (such as either structure shown in FIG. 14), andto select for receptors in which the indicator functions in the bindingof ligand. Hydrogen bonding or ionic substituents on the indicatorinvolved in analyte binding may have the capacity to change the electrondensity and/or rigidity of the indicator, thereby changing observablespectroscopic properties such as fluorescence quantum yield, maximumexcitation wavelength, maximum emission wavelength, and/or absorbance.This approach may not require the dissociation of a preloadedfluorescent ligand (limited in response time by k_(off)), and maymodulate the signal from essentially zero without analyte to largelevels in the presence of analyte.

In one embodiment, the microenvironment at the surface and interior ofthe resin beads may be conveniently monitored using spectroscopy whensimple pH sensitive dyes or solvachromic dyes are imbedded in the beads.As a guest binds, the local pH and dielectric constants of the beadschange, and the dyes respond in a predictable fashion. The binding oflarge analytes with high charge and hydrophobic surfaces, such as DNA,proteins, and steroids, should induce large changes in localmicroenvironment, thus leading to large and reproducible spectralchanges. This means that most any receptor can be attached to a resinbead that already has a dye attached, and that the bead becomes a sensorfor the particular analyte.

In one embodiment, a receptor may be covalently coupled to an indicator.The binding of the analyte may perturb the local microenvironment aroundthe receptor leading to a modulation of the absorbance or fluorescenceproperties of the sensor.

In one embodiment, receptors may be used immediately in a sensing modesimply by attaching the receptors to a bead that is already derivatizedwith a dye sensitive to its microenvironment. This is offers anadvantage over other signaling methods because the signaling protocolbecomes routine and does not have to be engineered; only the receptorsneed to be engineered. The ability to use several different dyes withthe same receptor, and the ability to have more than one dye on eachbead allows flexibility in the design of a sensing particle.

Changes in the local pH, local dielectric, or ionic strength, near afluorophore may result in a signal. A high positive charge in amicroenvironment leads to an increased pH since hydronium migrates awayfrom the positive region. Conversely, local negative charge decreasesthe microenvironment pH. Both changes result in a difference in theprotonation state of pH sensitive indicators present in thatmicroenvironment. Many common chromophores and fluorophores are pHsensitive. The interior of the bead may be acting much like the interiorof a cell, where the indicators should be sensitive to local pH.

The third optical transduction scheme involves fluorescence energytransfer. In this approach, two fluorescent monomers for signaling maybe mixed into a combinatorial split synthesis. Examples of thesemonomers are depicted in FIG. 25. Compound 1620 (a derivative offluorescein) contains a common colorimetric/fluorescent probe that maybe mixed into the oligomers as the reagent that will send out amodulated signal upon analyte binding. The modulation may be due toresonance energy transfer to monomer 1640 (a derivative of rhodamine).

When an analyte binds to the receptor, structural changes in thereceptor will alter the distance between the monomers (schematicallydepicted in FIG. 23, 1460 corresponds to monomer 1620 and 1480corresponds to monomer 1640). It is well known that excitation offluorescein may result in emission from rhodamine when these moleculesare oriented correctly. The efficiency of resonance energy transfer fromfluorescein to rhodamine will depend strongly upon the presence ofanalyte binding; thus, measurement of rhodamine fluorescence intensity(at a substantially longer wavelength than fluorescein fluorescence)will serve as an indicator of analyte binding. To greatly improve thelikelihood of a modulatory fluorescein-rhodamine interaction, multiplerhodamine tags can be attached at different sites along a combinatorialchain without substantially increasing background rhodamine fluorescence(only rhodamine very close to fluorescein will yield appreciablesignal). In one embodiment, depicted in FIG. 23, when no ligand ispresent, short wavelength excitation light (blue light) excites thefluorophore 1460, which fluoresces (green light). After binding ofanalyte ligand to the receptor, a structural change in the receptormolecule brings fluorophore 1460 and fluorophore 1480 in proximity,allowing excited-state fluorophore 1460 to transfer its energy tofluorophore 1480. This process, fluorescence resonance energy transfer,is extremely sensitive to small changes in the distance between dyemolecules (e.g., efficiency˜[distance]⁻⁶).

In another embodiment, photoinduced electron transfer (PET) may be usedto analyze the local microenvironment around the receptor. The methodsgenerally include a fluorescent dye and a fluorescence quencher. Afluorescence quencher is a molecule that absorbs the emitted radiationfrom a fluorescent molecule. The fluorescent dye, in its excited state,will typically absorbs light at a characteristic wavelength and thenre-emits the light at a characteristically different wavelength. Theemitted light, however, may be reduced by electron transfer with thefluorescent quencher, which results in quenching of the fluorescence.Therefore, if the presence of an analyte perturbs the quenchingproperties of the fluorescence quencher, a modulation of the fluorescentdye may be observed.

The above-described signaling methods may be incorporated into a varietyof receptor-indicator-polymeric resin systems. Turning to FIG. 24A, anindicator I and receptor R may be coupled to a polymeric resin. In theabsence of an analyte, the indicator may produce a signal in accordancewith the local microenvironment. The signal may be an absorbance at aspecific wavelength or fluorescence. When the receptor interacts with ananalyte, the local microenvironment may be altered such that theproduced signal is altered. In one embodiment, depicted in FIG. 24A, theindicator may partially bind to the receptor in the absence of analyteA. When the analyte is present, the indicator may be displaced from thereceptor by the analyte. The local microenvironment for the indicatortherefore changes from an environment where the indicator is bindingwith the receptor, to an environment where the indicator is no longerbound to the receptor. Such a change in environment may induce a changein the absorbance or fluorescence of the indicator.

In another embodiment, depicted in Turning to FIG. 24C, indicator I maybe coupled to receptor R. The receptor may be coupled to a polymericresin. In the absence of analyte A, the indicator may produce a signalin accordance with the local microenvironment. The signal may be anabsorbance at a specific wavelength or fluorescence. When the receptorinteracts with an analyte, the local microenvironment may be alteredsuch that the produced signal is altered. In contrast to the casedepicted in FIG. 24A, the change in local microenvironment may be due toa conformation change of the receptor due to the biding of the analyte.Such a change in environment may induce a change in the absorbance orfluorescence of the indicator.

In another embodiment, depicted in FIG. 24E, indicator I may be coupledto a receptor by a linker. The linker may have a sufficient length toallow the indicator to bind to the receptor in the absence of analyte A.Receptor R may be coupled to a polymeric resin. In the absence ofanalyte A, the indicator may produce a signal in accordance with thelocal microenvironment. As depicted in FIG. 24E, the indicator maypartially bind to the receptor in the absence of an analyte. When theanalyte is present, the indicator may be displaced from the receptor bythe analyte. The local microenvironment for the indicator thereforechanges from an environment where the indicator is binding with thereceptor, to an environment where the indicator is no longer bound tothe receptor. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

In another embodiment, depicted in FIG. 24H, receptor R may be coupledto a polymeric resin by a first linker. An indicator may be coupled tothe first linker. In the absence of analyte A, the indicator may producea signal in accordance with the local microenvironment. The signal maybe an absorbance at a specific wavelength or fluorescence. When thereceptor interacts with an analyte, the local microenvironment may bealtered such that the produced signal is altered. In one embodiment, asdepicted in FIG. 24H, the indicator may partially bind to the receptorin the absence of an analyte. When the analyte is present, the indicatormay be displaced from the receptor by the analyte. The localmicroenvironment for the indicator therefore changes from an environmentwhere the indicator is binding with the receptor, to an environmentwhere the indicator is no longer bound to the receptor. Such a change inenvironment may induce a change in the absorbance or fluorescence of theindicator.

In another embodiment, the use of fluorescence resonance energy transferor photoinduced electron transfer may be used to detect the presence ofan analyte. Both of these methodologies involve the use of twofluorescent molecules. Turning to FIG. 24B, a first fluorescentindicator B may be coupled to receptor R. Receptor R may be coupled to apolymeric resin. A second fluorescent indicator C may also be coupled tothe polymeric resin. In the absence of an analyte, the first and secondfluorescent indicators may be positioned such that fluorescence energytransfer may occur. In one embodiment, excitation of the firstfluorescent indicator may result in emission from the second fluorescentindicator when these molecules are oriented correctly. Alternatively,either the first or the second fluorescent indicator may be afluorescence quencher.

When the two indicators are properly aligned, the excitation of thefluorescent indicators may result in very little emission due toquenching of the emitted light by the fluorescence quencher. In bothcases, the receptor and indicators may be positioned such thatfluorescent energy transfer may occur in the absence of an analyte. Whenthe analyte is presence the orientation of the two indicators may bealtered such that the fluorescence energy transfer between the twoindicators is altered. In one embodiment, the presence of an analyte maycause the indicators to move further apart. This has an effect ofreducing the fluorescent energy transfer. If the two indicators interactto produce an emission signal in the absence of an analyte, the presenceof the analyte may cause a decrease in the emission signal.Alternatively, if one the indicators is a fluorescence quencher, thepresence of an analyte may disrupt the quenching and the fluorescentemission from the other indicator may increase. It should be understoodthat these effects will reverse if the presence of an analyte causes theindicators to move closer to each other.

In another embodiment, depicted in FIG. 24D, a first fluorescentindicator B may be coupled to receptor R. A second fluorescent indicatorC may also be coupled to the receptor. Receptor R may be coupled to apolymeric resin. In the absence of an analyte, the first and secondfluorescent indicators may be positioned such that fluorescence energytransfer may occur. In one embodiment, excitation of the firstfluorescent indicator may result in emission from the second fluorescentindicator when these molecules are oriented correctly. Alternatively,either the first or the second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, depicted inFIG. 24D, the presence of an analyte may cause the indicators to movefurther apart. This has an effect of reducing the fluorescent energytransfer. If the two indicators interact to produce an emission signalin the absence of an analyte, the presence of the analyte may cause adecrease in the emission signal. Alternatively, if one the indicators isa fluorescence quencher, the presence of an analyte may disrupt thequenching and the fluorescent emission from the other indicator mayincrease. It should be understood that these effects would reverse ifthe presence of an analyte causes the indicators to move closer to eachother.

In a similar embodiment to FIG. 24D, the first fluorescent indicator Band second fluorescent indicator C may be both coupled to receptor R, asdepicted in FIG. 24F. Receptor R may be coupled to a polymeric resin.First fluorescent indicator B may be coupled to receptor R by a linkergroup. The linker group may allow the first indicator to bind thereceptor, as depicted in FIG. 24F. In the absence of an analyte, thefirst and second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. When the analyte is presence,the first indicator may be displaced from the receptor, causing thefluorescence energy transfer between the two indicators to be altered.

In another embodiment, depicted in FIG. 24G, first fluorescent indicatorB may be coupled to a polymeric resin. Receptor R may also be coupled toa polymeric resin. A second fluorescent indicator C may be coupled tothe receptor R. In the absence of an analyte, the first and secondfluorescent indicators may be positioned such that fluorescence energytransfer may occur. In one embodiment, excitation of the firstfluorescent indicator may result in emission from the second fluorescentindicator when these molecules are oriented correctly. Alternatively,either the first or the second fluorescent indicator may be afluorescence quencher.

When the two indicators are properly aligned, the excitation of thefluorescent indicators may result in very little emission due toquenching of the emitted light by the fluorescence quencher. In bothcases, the receptor and indicators may be positioned such thatfluorescent energy transfer may occur in the absence of an analyte. Whenthe analyte is presence the orientation of the two indicators may bealtered such that the fluorescence energy transfer between the twoindicators is altered. In one embodiment, the presence of an analyte maycause the indicators to move further apart. This has an effect ofreducing the fluorescent energy transfer. If the two indicators interactto produce an emission signal in the absence of an analyte, the presenceof the analyte may cause a decrease in the emission signal.Alternatively, if one the indicators is a fluorescence quencher, thepresence of an analyte may disrupt the quenching and the fluorescentemission from the other indicator may increase. It should be understoodthat these effects would reverse if the presence of an analyte causesthe indicators to move closer to each other.

In another embodiment, depicted in FIG. 24I, a receptor R may be coupledto a polymeric resin by a first linker. First fluorescent indicator Bmay be coupled to the first linker. Second fluorescent indicator C maybe coupled to receptor R. In the absence of analyte A, the first andsecond fluorescent indicators may be positioned such that fluorescenceenergy transfer may occur. In one embodiment, excitation of the firstfluorescent indicator may result in emission from the second fluorescentindicator when these molecules are oriented correctly. Alternatively,either the first or the second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, the presenceof an analyte may cause the indicators to move further apart. This hasan effect of reducing the fluorescent energy transfer. If the twoindicators interact to produce an emission signal in the absence of ananalyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects would reverse if the presence of ananalyte causes the indicators to move closer to each other.

In one embodiment, polystyrene/polyethylene glycol resin beads may beused as a polymeric resin since they are highly water permeable, andgive fast response times to penetration by analytes. The beads may beobtained in sizes ranging from 5 microns to 250 microns. Analysis with aconfocal microscope reveals that these beads are segregated intopolystyrene and polyethylene glycol microdomains, at about a 1 to 1ratio. Using the volume of the beads and the reported loading of 300pmol/bead, we can calculate an average distance of 35 Å between terminalsites. This distance is well within the Forester radii for thefluorescent dyes that we are proposing to use in our fluorescenceresonance energy transfer (“FRET”) based signaling approaches. Thisdistance is also reasonable for communication between binding events andmicroenvironment changes around the fluorophores.

The derivatization of the beads with receptors and indicators may beaccomplished by coupling carboxylic acids and amines using EDC and HOBT.Typically, the efficiency of couplings are greater that 90% usingquantitative ninhydrin tests. (See Niikura, K.; Metzger, A.; and Anslyn,E. V. “A Sensing Ensemble with Selectivity for lositol Trisphosphate”,J. Am. Chem. Soc. 1998, 120, 0000, which is incorporated herein byreference). The level of derivatization of the beads is sufficient toallow the loading of a high enough level of indicators and receptors toyield successful assays. However, an even higher level of loading may beadvantageous since it would increase the multi-valency effect forbinding analytes within the interior of the beads. We may increase theloading level two fold and ensure that two amines are close in proximityby attaching an equivalent of lysine to the beads (see FIG. 26). Theamines may be kept in proximity so that binding of an analyte to thereceptor will influence the environment of a proximal indicator.

Even though a completely random attachment of indicator and a receptorlead to an effective sensing particle, it may be better to rationallyplace the indicator and receptor in proximity. In one embodiment, lysinethat has different protecting groups on the two different amines may beused, allowing the sequential attachment of an indicator and a receptor.If needed, additional rounds of derivatization of the beads with lysinemay increase the loading by powers of two, similar to the synthesis ofthe first few generations of dendrimers.

In contrast, too high a loading of fluorophores will lead toself-quenching, and the emission signals may actually decrease withhigher loadings. If self-quenching occurs for fluorophores on thecommercially available beads, the terminal amines may be incrementallycapped, thereby incrementally lowering loading of the indicators.

Moreover, there should be an optimum ratio of receptors to indicators.The optimum ratio is defined as the ratio of indicator to receptor togive the highest response level. Too few indicators compared toreceptors may lead to little change in spectroscopy since there will bemany receptors that are not in proximity to indicators. Too manyindicators relative to receptors may also lead to little change inspectroscopy since many of the indicators will not be near receptors,and hence a large number of the indicators will not experience a changein microenvironment. Through iterative testing, the optimum ratio may bedetermined for any receptor indicator system.

This iterative sequence will be discussed in detail for a particledesigned to signal the presence of an analyte in a fluid. The sequencebegins with the synthesis of several beads with different loadings ofthe receptor. The loading of any receptor may be quantitated using theninhydrin test. (The ninhydrin test is described in detail in Kaiser,E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. “Color Test forDetection of Free Terminal Amino Groups in the Solid-Phase Synthesis ofPeptides”, Anal. Biochem. 1970, 34, 595-598, which is incorporatedherein by reference). The number of free amines on the bead is measuredprior to and after derivatization with the receptor, the difference ofwhich gives the loading. Next, the beads undergo a similar analysis withvarying levels of molecular probes. The indicator loading may bequantitated by taking the absorption spectra of the beads. In thismanner, the absolute loading level and the ratio between the receptorand indicators may be adjusted. Creating calibration curves for theanalyte using the different beads will allow the optimum ratios to bedetermined.

The indicator loading may be quantitated by taking the absorptionspectra of a monolayer of the beads using our sandwich technique (SeeFIG. 27). The sandwich technique involves measuring the spectroscopy ofsingle monolayers of the beads. The beads may be sandwiched between twocover slips and gently rubbed together until a monolayer of the beads isformed. One cover slip is removed and meshed with dimensions on theorder of the beads is then place over the beads, and the cover slipreplaced. This sandwich is then placed within a cuvette, and theabsorbance or emission spectra are recorded. Alternatively, a sensorarray system, as described above, may be used to analyze the interactionof the beads with the analyte.

A variety of receptors may be coupled to the polymeric beads. Many ofthese receptors have been previously described. Other receptors areshown in FIG. 28.

As described generally above, an ensemble may be formed by a syntheticreceptor and a probe molecule, either mixed together in solution orbound together on a resin bead. The modulation of the spectroscopicproperties of the probe molecule results from perturbation of themicroenvironment of the probe, due to interaction of the receptor withthe analyte; often a simple pH effect. The use of a probe moleculecoupled to a common polymeric support may produce systems that givecolor changes upon analyte binding. A large number of dyes arecommercially available, many of which may be attached to the bead via asimple EDC/HOBT coupling (FIG. 29 shows some examples of indicators).These indicators are sensitive to pH, and respond to ionic strength andsolvent properties. When contacted with an analyte, the receptorinteracts with the analyte such that microenvironment of the polymericresin may become significantly changed. This change in themicroenvironment may induce a color change in the probe molecule. Thismay lead to an overall change in the appearance of the particleindicating the presence of the analyte.

Since many indicators are sensitive to pH and local ionic strength,index of refraction, and/or metal binding, lowering the local dielectricconstant near the indicators may modulate the activity of the indicatorssuch that they are more responsive. A high positive charge in amicroenvironment leads to an increased pH since hydronium ions migrateaway from the positive region. Conversely, local negative chargedecreases the microenvironment pH. Both changes result in a differenceon the protonation state of a pH sensitive indicator present in thatmicroenvironment. The altering of the local dielectric environment maybe produced by attaching molecules of differing dielectric constants tothe bead proximate to the probe molecules. Examples of molecules, whichmay be used to alter the local dielectric environment include, but arenot limited to, planar aromatics, long chain fatty acids, and oligomerictracts of phenylalanine, tyrosine, and tryptophan. Differing percentagesof these compounds may be attached to the polymeric bead to alter thelocal dielectric constant.

Competition assays may also be used to produce a signal to indicate thepresence of an analyte. The high specificity of antibodies makes themthe current tools of choice for the sensing and quantitation ofstructurally complex molecules in a mixture of analytes. These assaysrely on a competition approach in which the analyte is tagged and boundto the antibody. Addition of the untagged analyte results in a releaseof the tagged analytes and spectroscopic modulation is monitored.Surprisingly, although competition assays have been routinely used todetermine binding constants with synthetic receptors, very little workhas been done exploiting competition methods for the development ofsensors based upon synthetic receptors. Examples of the competitiveassay is described in U.S. Patent Application Publication No. US2002-0197622 A1, which is fully incorporated by reference as if fullyset forth herein.

Dramatic spectroscopy changes accompany the chelation of metals toligands that have chromophores. In fact, most colorimetric/fluorescentsensors for metals rely upon such a strategy. Binding of the metal tothe inner sphere of the ligand leads to ligand/metal charge transferbands in the absorbance spectra, and changes in the HOMO-LUMO gap thatleads to fluorescence modulations. Examples of spectroscopy changes fromthe chelation of metals to ligands is described in U.S. PatentApplication Publication No. US 2002-0197622 A1, which is fullyincorporated by reference as if fully set forth herein.

In one embodiment, an indicator may be coupled to a bead and further maybe bound to a receptor that is also coupled to the bead. Displacement ofthe indicator by an analyte will lead to signal modulation. Such asystem may also take advantage of fluorescent resonance energy transferto produce a signal in the presence of an analyte. Fluorescenceresonance energy transfer is a technique that can be used to shift thewavelength of emission from one position to another in fluorescencespectra. In the manner it creates, a much more sensitive assay since onecan monitor intensity at two wavelengths. The method involves theradiationless transfer of excitation energy from one fluorophore toanother. The transfer occurs via coupling of the oscillating dipoles ofthe donor with the transition dipole of the acceptor. The efficiency ofthe transfer is described by equations first derived by Forester. Theyinvolve a distance factor R, orientation factor k, solvent index ofrefraction N, and spectral overlap J.

In order to incorporate fluorescence resonance energy transfer into aparticle a receptor and two different indicators may be incorporatedonto a polymeric bead. In the absence of an analyte the fluorescenceresonance energy transfer may occur giving rise to a detectable signal.When an analyte interacts with a receptor, the spacing between theindicators may be altered. Altering this spacing may cause a change inthe fluorescence resonance energy transfer, and thus, a change in theintensity or wavelength of the signal produced. The fluorescenceresonance energy transfer efficiency is proportional to the distance Rbetween the two indicators by 1/R⁶. Thus, slight changes in the distancebetween the two indicators may induce significant changes in thefluorescence resonance energy transfer.

In one embodiment, various levels of coumarin and fluorescein may beloaded onto resin beads to achieve gradations in FRET levels from zeroto 100%. FIG. 30 shows a 70/30 ratio of emission from5-carboxyfluorescein and coumarin upon excitation of coumarin in varioussolvents. However, other solvents give dramatically different extents ofFRET. This shows that the changes in the interior of the beads do leadto a spectroscopic response. This data also shows that differentialassociation of the various solvents and 5-carboxyfluorescein on resinbeads as a function of solvents. This behavior is evoked from thesolvent association with the polymer itself, in the absence ofpurposefully added receptors. We may also add receptors, which exhibitstrong/selective association with strategic analytes. Such receptors mayinduce a modulation in the ratio of FRET upon analyte binding, withinthe microenvironment of the polystyrene/polyethylene glycol matrices.

In order to incorporate a wavelength shift into fluorescence assays,receptors 3-6 may be coupled to the courmarin/5-carboxyfluorescein beadspreviously discussed. When 5-carboxyfluorescein is bound to the variousreceptors and coumarin is excited, the emission will be primarily formcoumarin since the fluorescein will be bound to the receptors. Upondisplacement of the 5-carboxyfluorescein by the analytes, emissionshould shift more toward 5-carboxyfluorescein since it will be releasedto the bead environment, which possesses coumarin. This will give us awavelength shift in the fluorescence, which is inherently more sensitivethan the modulation of intensity at a signal wavelength.

There should be large changes in the distance between indicators R onthe resin beads. When the 5-carboxyfluorescein is bound, thedonor/acceptor pair should be farther than when displacement takesplace; the FRET efficiency scales as 1/R⁶. The coumarin may be coupledto the beads via a floppy linker, allowing it to adopt manyconformations with respect to a bound 5-carboxyfluorescein. Hence, it ishighly unlikely that the transition dipoles of the donor and acceptorwill be rigorously orthogonal.

Detection of polycarboxylic acids, tartrate, tetracycline amino acids,solvatochromic dyes, and ATP using fluorophores are described in U.S.Patent Application Publication No. US 2002-0197622 A1, which is inincorporated by reference as if fully set forth herein.

As described above, a particle, in some embodiments, possesses both theability to interact with the analyte of interest and to create amodulated signal. In one embodiment, the particle may include receptormolecules, which undergo a chemical change in the presence of theanalyte of interest. This chemical change may cause a modulation in thesignal produced by the particle. Chemical changes may include chemicalreactions between the analyte and the receptor. Receptors may includebiopolymers or organic molecules. Such chemical reactions may include,but are not limited to, cleavage reactions, oxidations, reductions,addition reactions, substitution reactions, elimination reactions, andradical reactions.

In one embodiment, the mode of action of the analyte on specificbiopolymers may be taken advantage of to produce an analyte detectionsystem. As used herein biopolymers refers to natural and unnatural:peptides, proteins, polynucleotides, and oligosaccharides. In someinstances, analytes, such as toxins and enzymes, will react withbiopolymer such that cleavage of the biopolymer occurs. In oneembodiment, this cleavage of the biopolymer may be used to produce adetectable signal. A particle may include a biopolymer and an indicatorcoupled to the biopolymer. In the presence of the analyte, thebiopolymer may be cleaved such that the portion of the biopolymer, whichincludes the indicator, may be cleaved from the particle. The signalproduced from the indicator is then displaced from the particle. Thesignal of the bead will therefore change thus indicating the presence ofa specific analyte.

Proteases represent a number of families of proteolytic enzymes thatcatalytically hydrolyze peptide bonds. Principal groups of proteasesinclude metalloproteases, serine porteases, cysteine proteases andaspartic proteases. Proteases, in particular serine proteases, areinvolved in a number of physiological processes such as bloodcoagulation, fertilization, inflammation, hormone production, the immuneresponse and fibrinolysis.

Numerous disease states are caused by and may be characterized byalterations in the activity of specific proteases and their inhibitors.For example, emphysema, arthritis, thrombosis, cancer metastasis andsome forms of hemophilia result from the lack of regulation of serineprotease activities. In case of viral infection, the presence of viralproteases has been identified in infected cells. Such viral proteasesinclude, for example, HIV protease associated with AIDS and NS3 proteaseassociated with Hepatitis C. Proteases have also been implicated incancer metastasis. For example, the increased presence of the proteaseurokinase has been correlated with an increased ability to metastasizein many cancers. Examples of detection of proteases is described in U.S.Patent Application Publication No. US 2002-0197622 A1, which isincorporated by reference as if fully set forth herein.

A variety of signaling mechanisms for the above described cleavagereactions may be used. In an embodiment, a fluorescent dye and afluorescence quencher may be coupled to the biopolymer on opposite sidesof the cleavage site. The fluorescent dye and the fluorescence quenchermay be positioned within the Förster energy transfer radius. The Försterenergy transfer radius is defined as the maximum distance between twomolecules in which at least a portion of the fluorescence energy emittedfrom one of the molecules is quenched by the other molecule. Försterenergy transfer has been described above. Before cleavage, little or nofluorescence may be generated by virtue of the molecular quencher. Aftercleavage, the dye and quencher are no longer maintained in proximity ofone another, and fluorescence may be detected (FIG. 31A). The use offluorescence quenching is described in U.S. Pat. No. 6,037,137, which isincorporated herein by reference. Further examples of this energytransfer are described in the following papers, all of which areincorporated herein by reference: James, T. D.; Samandumara, K. R. A.;Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995, 117, 8982. Murukami, H.;Nagasaki, T.; Hamachi, I.; Shinkai, S. Tetrahedron Lett., 34, 6273.Shinkai, S.; Tsukagohsi, K.; Ishikawa, Y.; Kunitake, T. J. Chem. Soc.Chem. Commun. 1991, 1039. Kondo, K.; Shiomi, Y.; Saisho, M.; Harada, T.;Shinkai, S. Tetrahedron. 1992, 48, 8239. Shiomi, Y.; Kondo, K.; Saisho,M.; Harada, T.; Tsukagoshi, K.; Shinkai, S. Supramol. Chem. 1993, 2, 11.Shiomni, Y.; Saisho, M.; Tsukagoshi, K.; Shinkai, S. J. Chem. Soc.Perkin Trans 1 1993, 2111. Deng, G.; James, T. D.; Shinkai, S. J. Am.Chem. Soc. 1994, 116, 4567. James, T. D.; Harada, T.; Shinkai, S. J.Chem. Soc. Chem. Commun. 1993, 857. James, T. D.; Murata, K.; Harada,T.; Ueda, K.; Shinkai, S. Chem. Lett. 1994, 273. Ludwig, R.; Harada, T.;Ueda, K.; James, T. D.; Shinkai, S. J. Chem. Soc. Perkin Trans 2. 1994,4, 497. Sandanayake, K. R. A. S.; Shinkai, S. J. Chem. Soc., Chem.Commun. 1994, 1083. Nagasaki, T.; Shinmori, H.; Shinkai, S. TetrahedronLett. 1994, 2201. Murakami, H.; Nagasaki, T.; Hamachi, I; Shinkai, S. J.Chem. Soc. Perkin Trans 2. 1994, 975. Nakashima, K.; Shinkai, S. Chem.Lett. 1994, 1267. Sandanayake, K. R. A. S.; Nakashima, K.; Shinkai, S.J. Chem. Soc. 1994, 1621. James, T. D.; Sandanayake, K. R. A. S.;Shinkai, S. J. Chem. Soc., Chem. Commun. 1994, 477. James, T. D.;Sandanayake, K. R. A. S.; Angew. Chem., Int. Ed. Eng. 1994, 33, 2207.James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature, 1995, 374,345.

The fluorophores may be linked to the peptide receptor by any of anumber of means well known to those of skill in the art. In anembodiment, the fluorophore may be linked directly from a reactive siteon the fluorophore to a reactive group on the peptide such as a terminalamino or carboxyl group, or to a reactive group on an amino acid sidechain such as a sulfur, an amino, a hydroxyl, or a carboxyl moiety. Manyfluorophores normally contain suitable reactive sites. Alternatively,the fluorophores may be derivatized to provide reactive sites forlinkage to another molecule. Fluorophores derivatized with functionalgroups for coupling to a second molecule are commercially available froma variety of manufacturers. The derivatization may be by a simplesubstitution of a group on the fluorophore itself, or may be byconjugation to a linker. Various linkers are well known to those ofskill in the art and are discussed below.

The fluorogenic protease indicators may be linked to a solid supportdirectly through the fluorophores or through the peptide backbonecomprising the indicator. In embodiments where the indicator is linkedto the solid support through the peptide backbone, the peptide backbonemay comprise an additional peptide spacer. The spacer may be present ateither the amino or carboxyl terminus of the peptide backbone and mayvary from about 1 to about 50 amino acids, preferably from 1 to about 20and more preferably from 1 to about 10 amino acids in length. The aminoacid composition of the peptide spacer is not critical as the spacerjust serves to separate the active components of the molecule from thesubstrate thereby preventing undesired interactions. However, the aminoacid composition of the spacer may be selected to provide amino acids(e.g. a cysteine or a lysine) having side chains to which a linker orthe solid support itself, is easily coupled. Alternatively, the linkeror the solid support itself may be attached to the amino terminus of orthe carboxyl terminus.

In an embodiment, the peptide spacer may be joined to the solid supportby a linker. The term “linker”, as used herein, refers to a moleculethat may be used to link a peptide to another molecule, (e.g. a solidsupport, fluorophore, etc.). A linker is a hetero or homobifunctionalmolecule that provides a first reactive site capable of forming acovalent linkage with the peptide and a second reactive site capable offorming a covalent linkage with a reactive group on the solid support.Linkers as use din these embodiments are the same as the previouslydescribed linkers.

In an embodiment, a first fluorescent dye and a second fluorescent dyemay be coupled to the biopolymer on opposite sides of the cleavage site.Before cleavage, a FRET (fluorescence resonance energy transfer) signalmay be observed as a long wavelength emission. After cleavage, thechange in the relative positions of the two dyes may cause a loss of theFRET signal and an increase in fluorescence from the shorter-wavelengthdye (FIG. 31B). Examples of solution phase FRET have been described inFörster , Th. “Transfer Mechanisms of Electronic Excitation:, Discuss.Faraday Soc., 1959, 27, 7; Khanna, P. L., Ullman, E. F.“4′,5′-Dimethoxyl-6-carboxyfluorescein: A novel dipole-dipole coupledfluorescence energy transfer acceptor useful for fluorescenceimmunoassays”, Anal. Biochem. 1980, 108, 156; and Morrison, L. E. “Timeresolved Detection of Energy Transfer: Theory and Application toImmunoassays”, Anal. Biochem. 1998, 174, 101, all of which areincorporated herein by reference.

In another embodiment, a single fluorescent dye may be coupled to thepeptide on the opposite side of the cleavage site to the polymericresin. Before cleavage, the dye is fluorescent, but is spatiallyconfined to the attachment site. After cleavage, the peptide fragmentcontaining the dye may diffuse from the attachment site (e.g., topositions elsewhere in the cavity) where it may be measured with aspatially sensitive detection approach, such as confocal microscopy(FIG. 31C). Alternatively, the solution in the cavities may be flushedfrom the system. A reduction in the fluorescence of the particle wouldindicate the presence of the analyte (e.g., a protease).

In another embodiment, a single indicator (e.g., a chromophore or afluorophore) may be coupled to the peptide receptor on the side of thecleavage site that remains on the polymeric resin or to the polymericresin at a location proximate to the receptor. Before cleavage, theindicator may produce a signal that reflects the microenvironmentdetermined by the interaction of the receptor with the indicator.Hydrogen bonding or ionic substituents on the indicator involved inanalyte binding have the capacity to change the electron density and/orrigidity of the indicator, thereby changing observable spectroscopicproperties such as fluorescence quantum yield, maximum excitationwavelength, or maximum emission wavelength for fluorophores orabsorption spectra for chromophores. When the peptide receptor iscleaved, the local pH and dielectric constants of the beads change, andthe indicator may respond in a predictable fashion. An advantage to thisapproach is that it does not require the dissociation of a preloadedfluorescent ligand (limited in response time by k_(off)). Furthermore,several different indicators may be used with the same receptor.Different beads may have the same receptors but different indicators,allowing for multiple testing for the presence of proteases.Alternatively, a single polymeric resin may include multiple dyes alongwith a single receptor. The interaction of each of these dyes with thereceptor may be monitored to determine the presence of the analyte.

The previously described sensor array systems may be used in diagnostictesting. Examples of diagnostic testing are described in U.S. PatentApplication Publication No. US 2002-0197622 A1, which is fullyincorporated herein by reference as if set forth herein.

In many common diagnostic tests, antibodies may be used to generate anantigen specific response. Generally, the antibodies may be produced byinjecting an antigen into an animal (e.g., a mouse, chicken, rabbit, orgoat) and allowing the animal to have an immune response to the antigen.Once an animal has begun producing antibodies to the antigen, theantibodies may be removed from the animal's bodily fluids, typically ananimal's blood (the serum or plasma) or from the animal's milk.Techniques for producing an immune response to antigens in animals arewell known.

Once removed from the animal, the antibody may be coupled to a polymericbead. The antibody may then act as a receptor for the antigen that wasintroduced into the animal. In this way, a variety of chemicallyspecific receptors may be produced and used for the formation of achemically sensitive particle. Once coupled to a particle, a number ofwell-known techniques may be used for the determination of the presenceof the antigen in a fluid sample. These techniques includeradioimmunoassay (RIA), microparticle capture enzyme immunoassay (MEIA),fluorescence polarization immunoassay (FPIA), and enzyme immunoassayssuch as enzyme-linked immunosorbent assay (ELISA). Immunoassay tests, asused herein, are tests that involve the coupling of an antibody to apolymeric bead for the detection of an analyte.

ELISA, FPIA and MEIA tests may typically involve the adsorption of anantibody onto a solid support. The antigen may be introduced and allowedto interact with the antibody. After the interaction is completed, achromogenic signal generating process may be performed which creates anoptically detectable signal if the antigen is present. Alternatively,the antigen may be bound to a solid support and a signal is generated ifthe antibody is present. Immunoassay techniques have been previouslydescribed, and are also described in the following U.S. Pat. Nos.3,843,696; 3,876,504; 3,709,868; 3,856,469; 4,902,630; 4,567,149 and5,681,754, all of which are incorporated by reference.

In ELISA testing, an antibody may be adsorbed onto a polymeric bead. Theantigen may be introduced to the assay and allowed to interact with anantibody for a period of hours or days. After the interaction iscomplete, the assay may be treated with a dye or stain, which reactswith the antibody. The excess dye may be removed through washing andtransferring of material. The detection limit and range for this assaymay be dependent on the technique of the operator.

Microparticle capture enzyme immunoassay (MEIA) may be used for thedetection of high molecular mass and low concentration analytes. TheMEIA system is based on increased reaction rate brought about with theuse of very small particles (e.g., 0.47 μm in diameter) as the solidphase. Efficient separation of bound from unbound material may becaptured by microparticles in a glass-fiber matrix. Detection limitsusing this type of assay are typically 50 ng/mL.

Fluorescence polarization immunoassay (FPIA) may be used for thedetection of low-molecular mass analytes, such as therapeutic drugs andhormones. In FPIA, the drug molecules from a patient serum and drugtracer molecules, labeled with fluorescein, compete for the limitedbinding sites of antibody molecules. With low patient drugconcentration, the greater number of binding sites may be occupied bythe tracer molecules. The reverse situation may apply for high patientdrug concentration. The extent of this binding may be measured byfluorescence polarization, governed by the dipolarity and fluorescentcapacity.

Cardiovascular risk factors may be predicted through the identificationof many different plasma-based factors using immunoassay. In oneembodiment, a sensor array may include one or more particles thatproduce a detectable signal in the presence of a cardiac risk factor. Insome embodiments, all of the particles in a sensor array may producedetectable signals in the presence of one or more cardiac risk factors.Particles disposed in a sensor array may use an immunoassay test todetermine the presence of cardiovascular risk factors.

As used herein, cardiovascular risk factors include any analytes thatcan be correlated to an increase or decrease in risk of cardiovasculardisease. Many different cardiovascular risk factors are know, includingproteins, organic molecules such as cholesterol and carbohydrates, andhormones. Serum lipids (e.g., HDL and LDL) and lipoproteins are thetraditional markers associated with cardiovascular disease. Studies,however, have demonstrated that serum lipids and lipoproteins predictless than half of future cardiovascular events and that other factorssuch as inflammation may contribute to coronary heart disease.Determining if an analyte is a risk factor for coronary heart diseasemay be achieved through analysis of the interrelationship betweenepidemiology and serum biomarker concentrations using risk factors.Examples of plasma based cardiovascular risk factors include, but arenot limited to, cytokines (e.g., interleukin-6), proteins (e.g.,C-reactive protein, lipoproteins, HDL, LDL, lipoprotein-a, VLDL, solubleintercellular adhesion molecule-1, fibrinogens, apolipoprotein A-1,apolipoprotein b), amino acids (e.g., homocysteine), bacteria (e.g.,Helicobacter pylori, chlamydia pneumoniae) and/or viruses (e.g., Herpesvirus hominis, cytomeglovirus).

Inflammation may contribute to the pathogenesis of arteriosclerosis bydestabilizing the fibrous cap of artheriosclerotic plaque causing plaquerupture. The destabilization may increase the risk of coronarythrombosis. The inflammatory process may be associated with increasedblood levels of cytokines and consequently, acute-phase reactants, suchas C-reactive protein (CRP). CRP is a circulating acute phase reactantthat reflects active systemic inflammation. Elevated plasma CRP levelsmay be associated with the extent and severity of arteriosclerosis thus,a higher risk for cardiovascular events. Numerous studies haveestablished CRP as a plasma-based strong risk predictor forcardiovascular disease in men and women. Plasma CRP levels may beassociated with the extent and severity of artheriosclerotic vasculardisease. In patients with known coronary artery disease, increasedlevels of CRP may be associated with an increased risk of futurecoronary events. CRP may be directly related to Interluekin-6 (IL-6)levels. IL-6 is a cytokine that may promote leukocyte adhesion to thevasculature. IL-6 may be a significant component of the inflammatoryprocess.

Soluble Intercellular Adhesion Molecule-1 (ICAM-1) may be another markerof inflammation associated with an increased risk for myocardialinfarction. ICAM-1 may mediate adhesion and transmigration of monocytesto the blood vessel wall. Fibrinogen, HDL, homocysteine, triglyceridesand CRP levels may be associated with ICAM-1 levels. ICAM-1 may beinvolved in endothelial cell activation and inflammation processes.ICAM-1 may also serve as a marker of early arteriosclerosis andassociated increase in chances for coronary artery disease.

Fibrinogen may mediate proartheriogenic effects by increasing plasmaviscosity, platelet aggregability, and by stimulating smooth muscle cellproliferation. In the study “European Concerted action on thrombosis anddisabilities Angina Pectoris Study Group”, Thompson, et al.; N. Engl. J.Med. 1995, pp. 635-611; high concentrations of fibrinogen and CRP werereported to associate with an increased risk for coronary disease. Highfibrinogen levels may be elevated, at least in part, because ofinflammatory changes that may occur with progressive arteriosclerosis.Once increased, fibrinogen may aggravate underlying vessel wall injuryand, by its procoagulant actions, predispose to further coronary events.In patients with chronic angina, fibrinogen levels may predictsubsequent acute coronary events. People with low fibrinogen levels mayhave a low risk of coronary events despite increased serum cholesterollevels. Therefore, fibrinogen may be used as a risk factor forartheriosclerotic vascular disease. Fibrinogen levels may be reduced bysmoking cessation, exercise, alcohol intake and estrogens. Fibrinogenlevels may increase with age, body size, diabetes, LDL-C, leukocytecount and menopause.

Studies have shown that increased levels of blood homocysteinerepresents an independent risk factor for acute coronary thrombosis, isa predictor of premature coronary disease/atherosclerosis, and isassociated with deep vein thrombosis and thromboembolism.

A number of studies have demonstrated elevated levels of the lipoproteinLp(a) in patients with angiographic evidence of coronary arterystenosis. As the blood Lp(a) level rises above normal, the odds ratiofor progression of CAD also rises, such that at greater than or equal to30 mg/dL, the risk is more than doubled. Other studies have relatedLp(a) levels to total cholesterol/HDL-cholesterol (TC/HDL-C) ratios suchthat when Lp(a) is greater than 50 mg/dL and the plasma TC/HDL-C ratiois greater than 5.8, the relative odds for CAD is 8.0-9.6.

Chlamydia pneumoniae, Helicobacter pylori and Herpesvirus hominis may beprimary etiologic factors or cofactors in the pathogenesis ofarteriosclerosis. The pathophysiological mechanisms by which infectiousagents may lead to arteriosclerosis may include, but are not limited to,production of proinflammatory mediators, stimulation of smooth muscleproliferation and endothelial dysfunction. Examples of proinflammatorymediators include but are not limited to, cytokines and free radicalspecies. Activation of an infectious organism within a chronic lesionmight lead to plaque inflammation, destabilization, and acute syndromes.Infection-induced inflammation may be amplified by outside factors (e.g.cigarette smoke) and so may be the risk for future cardiovascularevents.

Diagnostic testing of cardiovascular risk factors in humans may beperformed using a sensor array system customized for immunoassay. Thesensor array may include a variety of particles that are chemicallysensitive to a variety of cardiovascular risk factor analytes. In oneembodiment, the particles may be composed of polymeric beads. Attachedto the polymeric beads may be at least one receptor. The receptors maybe chosen based on its binding ability with the analyte of interest.(See FIG. 13)

The sensor array may be adapted for use with blood. Other body fluidssuch as, saliva, sweat, mucus, semen, urine and milk may also beanalyzed using a sensor array. The analysis of most bodily fluids,typically, will require filtration of the material prior to analysis.For example, cellular material and proteins may need to be removed fromthe bodily fluids. As previously described, the incorporation of filtersonto the sensor array platform, may allow the use of a sensor array withblood samples. These filters may also work in a similar manner withother bodily fluids, especially urine. Alternatively, a filter may beattached to a sample input port of the sensor array system, allowing thefiltration to take place as the sample is introduced into the sensorarray.

In an embodiment, cardiovascular risk factors may all be analyzed atsubstantially the same time using a sensor array system. The sensorarray may include all the necessary reagents and indicators required forthe visualization of each of these tests. In addition, the sensor arraymay be formed such that these reagents are compartmentalized. Forexample, the reagents required for an antigen test may be isolated fromthose for an antibody test. The sensor array may offer a completecardiovascular risk profile with a single test.

In an embodiment of a sensor array, particles may be selectivelyarranged in micromachined cavities localized on silicon wafers. Thecavities may be created with an anisotropic etching process as describedin U.S. Patent Application Publication No. US 2002-0197622 A1, which isfully incorporated herein by reference as if set forth herein. Thecavities may be pyramidal pit shaped with openings that allows for fluidflow through the cavity and analysis chamber and optical access.Identification and quantitation of the analytes may occur using acalorimetric and/or fluorescent change to a receptor and indicatormolecules that are covalently attached to termination sites on thepolymeric microspheres. Spectral data is extracted from the arrayefficiently using a charge-coupled device.

In an embodiment of a multiple receptor particle sensor array, differentantibody receptors may be coupled to different particles (see FIGS. 13and 14). The receptor bound particles may be placed in a sensor array asdescribed herein. A stream derived from a bodily fluid isolated from aperson may be passed over the array. The receptor specific analyte mayinteract with the different receptors. An enzyme linked proteinvisualization agent is added to the fluid phase. Chemical derivatizationof the visualization agent with a dye is performed. After binding to thebead-localized antibodies, the visualization agent reveals the presenceof complimentary antibodies at specific polymer bead sites. Level ofdetection of the antibodies concentration may be between about 1 and10,000 ng/mL. In an embodiment, the level of detection of the CRPantibodies concentration may be less than about 1 ng/mL.

In an embodiment, a mixture of visualization processes may be used. Forexample, the visualization process may include a protein conjugated witha fluorescent dye. A second visualization process may include a proteinconjugated with colloidal gold. The beads that are complexed withparticle-analyte-fluorescent dye signal generator may be visualizedthrough illumination at the excitation wavelength maximum of thefluorophore (e.g., 470 nm). Particle-analyte-colloidal gold conjugatedprotein may be visualized through exposure to a silver enhancersolution.

In an embodiment, a protein and a bacterium known to predictcardiovascular risk may be detected. For example, in a multiple receptorparticle sensor array, antibody receptors (e.g., CRP antibody, chlamydiapneumoniae antibody) may be coupled to different particles. The receptorbound particles may be placed in a sensor array. A stream containingmultiple analytes may be passed over the array. The receptor specificanalyte may interact with the CRP and/or chlamydia pneumonia boundantibodies. After the interaction is complete, a visualization agent maybe added to the sensor array. An optically detectable signal may bedetected, if the protein and/or bacterium is present. In an embodiment,the protein and bacterium receptors may be coupled to the same particle.

IL-6 regulates the production of CRP in acute phase inflammatoryresponse. Analysis of IL-6 and CRP in the blood serum may give a betterprediction of cardiovascular disease. In an embodiment, the analysis ofIL-6 and CRP in blood serum may be accomplished using a sensor array byincorporating particles that interact with CRP and IL-6. The intensityof the signal produced by the interaction of the particles with theanalytes may be used to determine the concentration of the CRP and IL-6in the blood serum. In some embodiments, multiple particles may be usedto detect, for example CRP. Each of the particles may produce a signalwhen a specific amount of CRP is present. If the CPR present is below apredetermined concentration, the particle may not produce a detectablesignal. By visually noting which of the particles are producing signalsand which are not, a semi-quantitative measure of the concentration ofCRP may be determined.

In an embodiment, the particles in the sensor array may be regenerated.A stream containing solutions (e.g., glycine-HCL buffer and/or MgCl₂,)efficient in releasing particle-analyte-visualization reagent complexmay be passed over the sensor array. Repetitive washings of theparticles in the array may be performed until an acceptable backgroundsignal using CCD methodology may be produced, in an embodiment. Thesensor array may then be treated with a stream of analyte solution,visualization receptor stream, then visualized using a reactant streamand/or fluorescence. Multiple cycles of testing and regeneration may beperformed with the same sensor array.

The use of a sensor array approach is an efficient, rapid andinexpensive analytical system. It has been customized for thesimultaneous detection of multiple cardiac risk factors in serum, suchas those associated with inflammation, which has recently beenidentified as a major contributor to the development of diseasedarteries. Inflammation of the arteries may explain heart disease inpeople without other known risk factors, such as people with normalcholesterol, low blood pressure and those in good physical shape. Thesepatients make up a third of all heart attack cases. The inflammationmarker C-reactive protein (CRP) is a target molecule a cardiac sensorarray because of its recognized importance as a strong predictor ofcardiac risk. Those individuals with high levels of CRP are twice aslikely as those with high cholesterol to die from heart attacks andstrokes.

Recent studies are finding an association between periodontal diseaseand heart disease. The most common of the periodontal diseases,gingivitis is an inflammation of the gingiva, or gums. Gingivitis occurswhen the bacteria, which exist normally in the oral cavity, multiply,increasing in mass and thickness until they form plaque. Plaque adheresto the surfaces of the teeth and adjacent gingiva and causes cellularinjury, with subsequent swelling and redness.

In one study, men with extensive gum disease (bleeding from every tooth)had over a fourfold greater risk for heart disease than men withoutperiodontal disease. The study also reported an association betweenstroke and gum disease. It is believed that in people withperiodontitis, normal oral activities, like brushing and chewing, cancause tiny injuries that release bacteria into the blood stream. Thebacteria that cause periodontitis may stimulate factors such as CRP andother proteins, which contribute to a higher risk for heart disease andstroke. In rare cases, periodontal bacteria can cause an infection inthe lining or valves of the heart called infective endocarditis.

Saliva may be used to provide instant, sensitive and accuratemeasurement of inflammatory markers indicative of generic disease. Inthis capacity, the herein described sensor array, and its customizedpanels, will be useful in both military and civilian settings requiringprompt identification of individuals that could potentially carry highlycommunicable diseases. Those sick patients identified by the “genericillness saliva test” may be subjected to more specific blood tests,which will identify the disease, and its causing agent.

In one embodiment, a sensor array may be used to address the need formeasuring inflammation markers such as CRP, IL-6 and TNF-α in saliva.Antibody reagents were evaluated for their capacity to capture anddetect each of the targeted analytes in a sandwich-type of immunoassayon a sensor array system. These tests involved coating agarose beadswith each of the antigen-capturing antibodies, exposing the beads to afixed concentration of antigen, and then to various fluorescentantigen-detecting antibodies. Those combinations of reagents thatproduced the strongest and most specific signal on the beads, wereclassified as matched pairs of antibodies that collectively provided themost efficient capture and detection of each analyte. Table 1 summarizesthe optimal reagents identified for ETC-based immunoassays for CRP, IL-6and TNF-α. The accompanying images demonstrate typical results obtainedfrom negative/control beads and beads sensitized for each of theanalytes.

TABLE 1 Matched Pairs of Antibodies for ETC-based Assays for CRP, IL-6and TNF-α Analyte Capturing Antibody Detecting Antibody C-ReactiveRabbit anti-CRP (Accurate Rabbit anti-CRP-Alexa- Protein Chem. Co.) 488(Accurate Chem. Co.) Interleukin-6 Mouse anti-IL-6 (R&D Mouse anti-IL-6-Biosystems) phycoerythrin (Diaclone- Cell Sciences) Tumor Necrosis Mouseanti-TNF-α Rabbit anti-TNF-α-FITC Factor -α (Biosource International)(Sigma Chemical Co.)

Once the optimal reagents were identified, conditions such as blockingsteps, capturing and detecting antibody concentrations andantigen/detecting antibody incubation times were explored to developquick and accurate chip-based assays capable of detecting analytes attheir reported physiological and patho-physiological levels.

A dose response for the CRP assay was created using CRP standardsdiluted in various matrices. Beads coated with 9 mg/nl of CRP-capturingantibody were utilized to detect various concentrations of CRP in a10-minute assay. Here, the matrix diluent determines the detection rangeof the assay. These studies identified phosphate buffered saline (PBS)as the diluent of choice for saliva CRP testing as it allows detectiondown to 10 fg/ml of CRP. A graph of various diluents compared todetection level of CRP is depicted in FIG. 32. Reference numbers 1660,1680, 1700, and 1720 indicate results with 0.1% BSAPBS, CRSP deplsaliva, APPCO diluent, and PBS, respectively.

A comparison between the commercially available High Sensitivity Assayfor CRP (based on ELISA methodology) and a sensor array counterpartclearly demonstrates the advantages of the herein described approach.The 10-minute sensor array assay may detect down to 10 fg/ml of CRPwhile the lowest detectable level of CRP with the 4-hour ELISA test isat 1.9 ng/ml. The sensor array-based test allows up to a 1000-folddilution of the viscous saliva sample, (as depicted in FIG. 33) therebyeliminating any matrix effects on the assay, while still maintaining adetection range between 10-10,000 pg/ml of CRP. A graph of thesensitivity of an ELISA test with respect to concentration of CRP isdepicted in FIG. 34, with the dotted lines indicating a useful rangebetween 1.9 and 47.3 ng/ml CRP.

Both ELISA and sensor based array approaches were then employed tomeasure CRP in saliva and serum samples in order to validate the system.However, since the ELISA method was not sensitive enough for thedetection of CRP in saliva, our validation studies ultimately involvedin parallel-testing of serum samples with both ELISA and sensor arraysystems. These results are depicted in FIGS. 35A-B, with referencenumbers 1740 and 1760 referring to ETC and ELISA, respectively.

These results clearly demonstrate that the sensor array approach isconsistent with ELISA, the current gold standard for immunoassays.Validation of the sensor array approach for saliva testing was alsoachieved by performing recovery studies in saliva samples. When salivasamples were spiked with known amounts of CRP the ETC reported valueswith 90-96% recovery rate (data not shown).

Both ELISA and sensor array based CRP assays were then employed for themeasurement of CRP in saliva samples from 3 groups of people: healthy,periodontal disease and edentulous. The traditional ELISA method wascapable of detecting CRP levels only from the periodontal group (datanot shown), presumably because of its lack in sensitivity. On thecontrary, and as shown in FIG. 36, the sensor array approach was capableof detecting and distinguishing levels of CRP from individuals from all3 groups. These data are summarized in FIG. 37. Additionally, matchedpairs of serum and saliva samples obtained from healthy individuals weremeasured for CRP using the sensor array approach. Even though the numberof samples tested was relatively low, these preliminary data suggestthat there is some correlation between CRP levels in saliva and serum(depicted in FIGS. 38A-B, with outliers excluded from FIG. 38B).

In an embodiment, different particles were manufactured by coupling adifferent antibody to an agarose bead particle. The agarose beadparticles were obtained from XC Corporation, Lowell Mass. The particleshad an average diameter of about 280 μm. The receptor ligands of theantibodies were attached to agarose bead particles using a reductiveamination process between a terminal resin bound gloyoxal and anantibody to form a reversible Schiff Base complex which can beselectively reduced and stabilized as covalent linkages by using areducing agent such as sodium cyanoborohydride. (See Borch et al. J. Am.Chem. Soc. 1971, 93, 2897-2904, which is incorporated fully herein.).

Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex were performedcalorimetrically using a CCD device, as previously described. Foridentification and quantification of the analyte species, changes in thelight absorption and light emission properties of the immobilizedparticle-analyte-visualization reagent complex were exploited.Identification based upon absorption properties are described herein.Upon exposure to the chromogenic signal generating process, colorchanges for the particles were about 90% complete within about one hourof exposure. Data streams composed of red, green, and blue (RGB) lightintensities were acquired and processed for each of the individualparticle elements.

In an embodiment, three different particles were manufactured bycoupling a HIV gp41/120, Influenza A and Hepatitis B (HBsAg) antigens toa bead particle (FIG. 39A). A series of HIV gp41/120 particles wereplaced within micromachined wells in a column of a sensor array.Similarly, Influenza A and Hepatitis B HBsAg particles are placed withinmicromachined wells of the sensor array. Introduction of a fluidcontaining HBsAg specific IgG was accomplished through the top of thesensor array with passage through the openings at the bottom of eachcavity. Unbound HBsAg-IgG was washed away using a pH 7.6 TRIS buffersolution. The particle-analyte complex was then exposed to a fluorophorevisualization reagent (e.g., CY2, FIG. 39B). A wash fluid was passedover the sensor array to remove the unreacted visualization agent.Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcalorimetrically using a CCD device. Particles that have form complexeswith HBsAg specific IgG exhibit a higher fluorescent value than thenoncomplexed Influenza A and HIV gp41/120 particles.

In an embodiment, a series of 10 particles were manufactured by couplinga CRP antibody to the particles at a high concentration (6 mg/mL). Asecond series of 10 particles were manufactured by coupling the CRPantibody to the particles at medium concentration (3 mg/mL). A thirdseries of 10 particles were manufactured by coupling the CRP antibody toparticles at a low concentration (0.5 mg/mL). A fourth series of 5particles were manufactured by coupling an immunoglobulin to theparticles. The fourth series of particles were a control for the assay.The particles were positioned in columns within micromachined wellsformed in silicon/silicon nitride wafers, thus confining the particlesto individually addressable positions on a multi-component chip.

The sensor array was blocked with 3% bovine serum albumin in phosphatebuffered solution (PBS) was passed through the sensor array system.Introduction of the analyte fluid (1,000 ng/mL of CRP) was accomplishedthrough the top of the sensor array with passage through the openings atthe bottom of each cavity. The particle-analyte complex was then exposedto a visualization reagent (e.g., horseradish peroxidase-linkedantibodies). A dye (e.g., 3-amino-9-ethylcarbazole) was added to thesensor array. Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcolorimetrically using a CCD device. The average blue responses of theparticles to CRP are depicted in FIG. 40. The particles with the highestconcentration of CRP-specific antibody (6 mg/mL) exhibited a darker bluecolor. The control particles (0 mg/mL) exhibited little color.

In an embodiment, a series of 10 particles were manufactured by couplinga CRP antibody to the particles at a high concentration (6 mg/mL). Asecond series of 10 particles were manufactured by coupling the CRPantibody to the particles at a medium concentration (3 mg/mL). A thirdseries of 10 particles were manufactured by coupling the CRP antibody tothe particles at a low concentration (0.5 mg/mL). A fourth series of 5particles were manufactured by coupling an immunoglobulin to theparticles. The fourth series of particles were a control for the assay.The particles were positioned in columns within micromachined wellsformed in silicon/silicon nitride wafers, thus confining the particlesto individually addressable positions on a multi-component chip.

The sensor array was blocked with 3% bovine serum albumin in phosphatebuffered solution (PBS) was passed through the sensor array system.Introduction of multiple streams of analyte fluids at varyingconcentrations (0 to 10,000 ng/mL) were accomplished through the top ofthe sensor array with passage through the openings at the bottom of eachcavity. The particle-analyte complex was then exposed to a visualizationreagent (e.g., horseradish peroxidase-linked antibodies). A dye (e.g.,3-amino-9-ethylcarbazole) was added to the sensor array.Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcalorimetrically using a CCD device. The dose dependent signals aregraphically depicted in FIG. 41.

In an embodiment, three different particles were manufactured bycoupling Fibrinogen. CRP and IL-6 antibodies to an agarose beadparticle. A series of CRP and IL-6 antibodies receptor particles, werepositioned within micromachined wells formed in silicon/silicon nitridewafers, thus confining the particles to individually addressablepositions on a multi-component chip. A series of control particles werealso placed in the sensor array. The sensor array was blocked by passing3% bovine serum albumin in phosphate buffered solution (PBS) through thesensor array system. Introduction of the analyte fluids was accomplishedthrough the top of the sensor array with passage through the openings atthe bottom of each cavity. The particle-analyte complex was then exposedto a visualization reagent (e.g., horseradish peroxidase-linkedantibodies). A dye (e.g., 3-amino-9-ethylcarbazole) was added to thesensor array. Spectrophotometric assays to probe for the presence of theparticle-analyte-visualization reagent complex was performedcalorimetrically using a CCD device. The average blue responses of theparticles to a fluid that includes buffer only (FIG. 42A), CRP (FIG.42B), interluekin-6 (FIG. 42C) and a combination of CRP andinterleukin-6 (FIG. 42D) are graphically depicted in FIGS. 42A-D.

This example demonstrated a number of important factors related to thedesign, testing, and functionality of micromachined array sensors forcardiac risk factor analyses. First, derivatization of agarose particleswith both antibodies was completed. These structures were shown to beresponsive to plasma and a visualization process. Second, response timeswell under one hour was found for colorimetric analysis. Third,micromachined arrays suitable both for confinement of particles, as wellas optical characterization of the particles, have been prepared.Fourth, each bead is a full assay, which allows for simultaneousexecution of multiple trials. More trials provide results that are moreaccurate. Finally, simultaneous detection of several analytes in amixture was made possible by analysis of the blue color patterns createdby the sensor array.

In an embodiment, 35 particles were manufactured by coupling a CRPantibody to the particles. The particles were positioned in columnswithin micromachined wells formed in silicon/silicon nitride wafers,thus confining the particles to individually addressable positions on amulti-component chip.

Beads coupled to 3 mg of antibody/ml of beads of either rabbitCRP-specific capture antibody (CRP) or an irrelevant rabbit anti-H.pylori-specific antibody (CTL) are tested for their capacity to detect1,000 ng/ml of CRP in human serum in continuous repetitive runs. FIG. 43depicts data collected using a colorimetric method. Here each cycleinvolves: i) injection of 1,000 ng/ml CRP, ii) addition ofHRP-conjugated anti-CRP detecting antibody, iii) addition of AEC, iv)elution of signal with 80% methanol, v) wash with PBS, vi) regenerationwith glycine-HCl buffer and vii) equilibration with PBS. Results shownin FIG. 43 are for the mean blue absorbance values. The results showthat regeneration of the system can be achieved over to allow multipletesting cycles to be performed with a single sensor array.

Several home testing kits have been developed for cardiac risk factorsthat rely on the use of an enzyme based testing. These types of testsare well suited to be incorporated as sensor array diagnotistic testingsystem.

Cholesterol, a common constituent of blood, is cardiac risk factor thatis frequently monitored by people. A number of home testing kits havebeen developed that rely on the use of an enzyme based testing methodfor the determination of the amount of cholesterol in blood. A methodfor the determination of cholesterol in blood is described in U.S. Pat.No. 4,378,429, which is incorporated by reference. The assay used inthis test may be adapted to use in a bead based sensor array system foranalysis of cardiac risk factors.

The triglyceride level in blood is also commonly tested for because itis an indicator of obesity, diabetes, and heart disease. A system forassaying for triglycerides in bodily fluids is described in U.S. Pat.No. 4,245,041, which is incorporated by reference. The assay used inthis test may be adapted to use in a bead based sensor array system foranalysis of cardiac risk factors.

The concentration of homocysteine may be an important indicator ofcardiovascular disease and various other diseases and disorders. Varioustests have been constructed to measure the concentration of homocysteinein bodily fluids. A method for the determination of homocysteine inblood, plasma, and urine is described in U.S. Pat. No. 6,063,581 andU.S. Pat. No. 5,478,729 entitled “Immunoassay for Homocysteine”, whichis incorporated by reference. The assay used in this test may be adaptedto use in a bead based sensor array system for analysis of cardiac riskfactors.

Cholesterol, triglyceride, homocysteine, and glucose testing may beperformed simultaneously using a sensor array system. Particles that aresensitive to cholesterol, triglyceride, homocysteine, or glucose may beplaced in the sensor array. Blood serum passed over the array may beanalyzed for glucose, triglyceride, and cholesterol. A key feature of aglucose, triglyceride, homocysteine, and/or cholesterol test is that thetest should be able to reveal the concentration of these compounds in aperson's blood. This may be accomplished using the sensor array bycalibrating the reaction of the particles to cholesterol, triglyceride,or glucose. The intensity of the signal may be directly correlated tothe concentration. In another embodiment, multiple particles may be usedto detect, for example, glucose. Each of the particles may produce asignal when a specific amount of glucose is present. If the glucosepresent is below a predetermined concentration, the particle may notproduce a detectable signal. By visually noting which of the particlesare producing signals and which are not, a semi-quantitative measure ofthe concentration of glucose may be determined. A similar methodologymay be used for cholesterol, triglyceride, homocysteine, or anycombination thereof (e.g.,glucose/cholesterol/triglyceride/homocysteine, cholesterol/triglyceride,glucose/triglyceride, glucose/cholesterol, etc.).

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1. A method for detecting one or more cardiovascular risk factoranalytes in saliva comprising: passing saliva over a sensor array, thesensor array comprising: a supporting member comprising a plurality ofcavities formed within the supporting member; a plurality of particles,at least one of the particles positioned in at least one of thecavities, wherein at least one of the particles is configured to producea signal in the presence of at least one of the cardiovascular riskfactor analytes during use, wherein at least one of the particles isconfigured to (a) produce a signal in the presence of C-reactive proteinand (b) exhibit a sensitivity sufficient for detecting C-reactiveprotein levels in saliva of less than 1.9 ng/ml; and monitoring aspectroscopic change of at least one of the particles as the saliva ispassed over the sensor array.
 2. The method of claim 1, wherein thesensor array further comprises a bottom layer and a cover, wherein thebottom layer is coupled to a bottom surface of the supporting member,and wherein the cover is coupled to a top surface of the supportingmember; and wherein both the bottom layer and the cover are coupled tothe supporting member such that at least one of the particles issubstantially contained in at least one of the cavities by the bottomlayer and the cover, and wherein the bottom layer and the cover aresubstantially transparent to light produced by a light source.
 3. Themethod of claim 1, wherein the sensor array further comprises a cover,the cover being coupled to the supporting member such that at least oneof the particles is substantially contained in at least one of thecavities by the cover, and wherein the cover is configured to allow thesaliva to pass through the cover to at least one of the particles, andwherein the supporting member and the cover are substantiallytransparent to light produced by a light source.
 4. The method of claim1, wherein the sensor array further comprises a cover positioned at adistance above the upper surface of the supporting member such that anopening is formed between the supporting member and the cover to allowthe saliva to enter at least one of the cavities via the opening, andwherein the cover inhibits dislodgment of at least one of the particlesfrom at least one of the cavities during use.
 5. The method of claim 1,wherein at least one of the cavities is configured such that the salivaentering the cavity passes through the supporting member during use. 6.The method of claim 1, wherein at least one of the cavities issubstantially tapered such that the width of the cavity narrows in adirection from a top surface of the supporting member toward a bottomsurface of the supporting member, and wherein a minimum width of thecavity is substantially less than a width of at least one of theparticles.
 7. The method of claim 1, wherein at least one of theparticles comprises a receptor molecule coupled to a polymeric resin. 8.The method of claim 1, wherein at least one of the particles comprises areceptor molecule coupled to a polymeric resin, and wherein the receptormolecule comprises a peptide.
 9. The method of claim 1, wherein at leastone of the particles comprises a receptor molecule coupled to apolymeric resin, and wherein the receptor molecule comprises a syntheticreceptor.
 10. The method of claim 1, wherein at least one of theparticles comprises a receptor molecule coupled to a polymeric resin,and wherein the receptor molecule comprises an antibody.
 11. The methodof claim 1, wherein at least a portion of the particle comprises areceptor molecule coupled to a polymeric resin, and wherein the receptormolecule comprises an antigen.
 12. The method of claim 1, wherein atleast one of the cardiovascular risk factor analytes is interleukin-6.13. The method of claim 1, wherein at least one of the cardiovascularrisk factor analytes is high density lipoprotein, low densitylipoprotein, very low density lipoprotein, cholesterol, interleukin-6,intercellular adhesion molecule-1, fibrinogen, homocysteine, folate,calcium, lipoprotein a, apolipoprotein A-1, apolipoprotein B,Helicobacter pylon, chlamydia pneumoniae, Herpes virus hominis, orcytomegalovirus.
 14. The method of claim 1, further comprisingsimultaneously detecting the presence of two or more cardiovascular riskfactor analytes in saliva.
 15. The method of claim 1, further comprisingsimultaneously detecting the presence of two or more cardiovascular riskfactor analytes in saliva, wherein one of the cardiovascular risk factoranalytes is high density lipoprotein, low density lipoprotein, very lowdensity lipoprotein, cholesterol, interleukin-6, intercellular adhesionmolecule-1, fibrinogen, homocysteine, folate, calcium, lipoprotein a,apolipoprotein A-1, apolipoprotein B, Helicobacter pylon, chlamydiapneumoniae, Herpes virus hominis, or cytomegalovirus.
 16. The method ofclaim 1, wherein two or more of the particles are configured to producea detectable signal in the presence of one or more cardiac risk factoranalytes.
 17. The method of claim 1, wherein the supporting membercomprises silicon.
 18. The method of claim 1, wherein the sensor arrayfurther comprises channels in the supporting member, wherein thechannels are configured to allow the saliva to flow through the channelsinto and away from at least one of the cavities.
 19. The method of claim1, wherein the sensor array further comprises a pump coupled to thesupporting member, wherein the pump is configured to direct the salivatowards at least one of the cavities, and wherein a channel is formed inthe supporting member, the channel coupling the pump to at least one ofthe cavities such that the saliva flows through the channel to at leastone of the cavities during use.
 20. A method for detecting two or morecardiovascular risk factor analytes in saliva comprising: passing salivaover a sensor array, the sensor array comprising: a supporting membercomprising two or more cavities formed within the supporting member; andtwo or more particles, at least a first one of the particles positionedin a first one of the cavities and a second one of the particlespositioned in a second one of the cavities, wherein at least the firstone of the particles is configured to (a) produce a signal in thepresence of C-reactive protein during use and (b) exhibit a sensitivitysufficient for detecting C-reactive protein levels in saliva of lessthan 1.9 ng/ml, and wherein at least the second one of the particles isconfigured to produce a signal in the presence of a cardiovascular riskfactor analyte different than C-reactive protein during use; andmonitoring a spectroscopic change of at least one of the particles asthe saliva is passed over the sensor array.